Thermodynamic and Kinetic Aspects of Hen Egg White ...

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November 2017

Thermodynamic and Kinetic Aspects of Hen EggWhite Lysozyme Amyloid AssemblyTatiana MitiUniversity of South Florida tmitimailusfedu

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Scholar Commons CitationMiti Tatiana Thermodynamic and Kinetic Aspects of Hen Egg White Lysozyme Amyloid Assembly (2017) Graduate Theses andDissertationshttpsscholarcommonsusfeduetd7425

Thermodynamic and Kinetic Aspects of Hen Egg White Lysozyme Amyloid Assembly

by

Tatiana Miti

A dissertation submitted in partial fulfillment

of the requirements for a degree of

Doctor of Philosophy

Department of Physics

College of Arts and Sciences

University of South Florida

Major Professor Martin Muschol PhD

Vladimir Uversky PhD

Sagar Pandit PhD

Ghanim Ullah PhD

Robert Hoy PhD

Piyush Koria PhD

Date of Approval

June 27 2017

Keywords Phase Diagram Nucleated Polymerization Nucleated Conformational Conversion

Copyright copy 2017 Tatiana Miti

ACKNOWLEDGMENTS

I would like to take this opportunity to thank all those who have supported and inspired

me during my dissertation research First and foremost I would like to thank my PhD advisor

Dr Martin Muschol for his direction inspiration knowledge and patience The fortuity to work

with him during my undergraduate years helped me discover my love for physics and played a

central role in my decision to continue doctoral research under his guidance His high ethical and

quality standards have made me a better experimentalist and this dissertation work would not

have been possible without his direction

I would also like to thank Dr Vladimir Uversky for his support throughout my PhD years His

advice and insights have helped me to take better decision in both my academic and non-

academic life I also tank Dr Leo Breydo for his great help and advice I thank both Dr Uversky

and Breydo for using their equipment and laboratory space in numerous occasions I am deeply

grateful Dr Sagar Pandit and Dr Robert Hoy for inspiring and helping me to explore the

theoretical and computational side of biophysics and soft matter

I would like to thank Dr Piyush Koria for the opportunity to explore both the biological

and the physical the realm of synthetic peptides The two years of collaboration have given me

the opportunity learn new experimental skills and gain knowledge in a different area of

biophysics

I thank Dr Ghanim Ullah for his great help in modeling our amyloid aggregation

behavior During our collaboration so far we gained new insights into the molecular

mechanisms of amyloid aggregation as well as point out some limitations or unexplored

experimental approaches

I thank Dr Garrett Matthews for allowing me to use his AFM and Himanshu Verma for

training me

I would like to give special thanks to my former lab member Dr Shannon Hill for her

experimental training advice encouragement and friendship during my first years in the

laboratory She has been and continues to be a great inspiration for me

I would like to thank my former lab colleague Mentor Mulaj for collecting a part of

AFM images presented in this dissertation and also for his support and help in the lab I would

like to thank my former lab colleague Joseph Foley for collecting a part of FTIR images

presented in this dissertation and also for his kindness support and help in the lab

I am especially grateful for Chamani Nyangoda and Jeremy Barton who have become

great friends and a source of inspiration The lab would not be the same without your cheerful

presence

I am thankful for my fellow friends in the physics department Alan Kramer and Tatiana

Eggers for their emotional support and encouragement

I am endlessly grateful to my mother and sister for their support love and patience during

my university years They have given me the strength and inspiration to continue despite all

adversity

Last but not least I would like to say thank you to my wonderful husband Mikalai He

has been my best friend my pillar and my source of optimism and calm during the last five

years I could have not done this without him

i

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vi

I Introduction 1

I1 Amyloidosis as Health Care Challenge helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip1

I2 Structural Hallmarks of ―Classical Amyloid Fibrils 3

I3 Amyloid Aggregation Pathways 6

I4 Amyloid Aggregation Mechanisms 7

I5 Amyloid Species Phase Diagram(s) and Their Importance in the Field 10

I6 Motivation and Goals for My Research 12

II Methods and Experimental Protocols 14

II1 Materials and Sample Preparation Protocols 14

II11 Fresh Hen Egg White Lysozyme Sample Preparation 14

II12 Thioflavin T Stock Preparation 15

II13 Rigid Fibrils and Curvilinear Fibrils Aggregates for Isolation 15

II14 Rigid Fibrils Isolation Protocol 16

II15 Curvilinear Fibril Decay Experiment 17

II16 Determination of Rigid Fibrils Nucleation Lag Periods 17

II2 Methods and Measurements Parameters 19

II21 Static and Dynamic Light Scattering Kinetics Measurements 20

II22 Thioflavin T Fluorescence Monitored Hen Egg White Lysozyme

Amyloid Fibril Formation Kinetics 20

II23 Fourier Transform Infrared Spectroscopy Measurements 22

II24 Atomic Forces Microscopy Imaging 25

II25 Circular Dischroism Spectroscopy Measurements 25

II26 Gel Electrophoresis Experiment 26

III Hen Egg Lysozyme Model 27

III1 Hen Egg White Lysozyme General Biophysical Characterization 27

III2 Hen Egg White Lysozyme Link to Amyloid Diseases 29

IV Results 32

IV1 Hen Egg Lysozymelsquos Two Assembly Pathways Kinetics and Aggregates

Morphologies 32

ii

IV2 Hen Egg Lysozyme Aggregateslsquo Structures and Their Time Evolution 38

IV21 Probing Hen Egg Lysozyme Aggregateslsquo Structures and Their

Time Evolution Using Fourier Transform Infrared Spectroscopy 38

IV22 Probing Hen Egg Lysozyme Aggregateslsquo Structures Time

Evolution Using Thioflavin T 41 IV23 Investigation of Hydrolysis Effects on Thioflavin T Aggregate -

Specific Response 44 IV3 Hen Egg Lysozyme Amyloid Aggregates Kinetic Phase Diagram and

Critical Oligomer Concentration (COC) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46

IV31 Mapping the Hen Egg White Lysozyme Amyloid Phase Diagram 46

IV32 Amyloid Oligomer and Curvilinear Fibrils Share a Common

Transition Boundary 49

IV33 Amyloid Oligomers and Their Curvilinear Fibrils Are

Thermodynamically Metastable 50

IV34 Precipitation Boundary Structural Analysis via Infrared

Spectroscopy 51

IV35 Model of Colloidal Charge Repulsion Reproduces Critical

Oligomer Concentration Boundary 53

IV4 Hen Egg Lysozyme Rigid Fibrils Nucleation Mechanisms Above and

Below the Critical Oligomer Concentration 54

IV41 Amyloid Nucleation and Growth Kinetics Reflect Basic

Aspects of Amyloid Phase Diagram 55

IV42 Amyloid Phase Diagram Permits Unbiased Test of On-

Pathway Nucleated Conformational Conversion vs Competitive

Nucleated Polymerization 59

V ConclusionsDiscussion 66

References helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip71

Appendices 89

Appendix A Oligomer and Precipitate Solubility Boundary Theoretical Model 89

A1 Model of Colloidal Charge Repulsion Reproduces Critical Oligomer

Concentration Boundary 89

A2 Loss of Colloidal Stability Predicts Onset of Oligomer Precipitation 92

Appendix B Methods Theoretical Background 95

B1 Static and Dynamic Light Scattering Theoretical Background helliphelliphellip95

B2 Thioflavin T Fluorescence Theoretical Background 102

B3 Protein Fourier Transform Infrared Spectroscopy Theoretical

Background 104

B4 Atomic Force Microscopy Theoretical Background 107

B5 Circular Dichroism Spectroscopy Theoretical Background 110

B6 Gel Electrophoresis Theoretical Background 111

Appendix C Copyright Permissions 112

Appendix References 120

iii

LIST OF TABLES

Table 1 Amyloid Diseases and Its Associated Polypeptides or Proteins 2

Table 2 Amide I Band Assignment to Protein Secondary Structure Motifs

Empirical vs Theoretical Values 23

Table 3 Comparison of Native vs Amyloid Fibrils Core β-sheets 24

Table 4 Summary of Rigid and OligomersCurvilinear Fibrils Morphologies

Determined by Atomic Forces Microscopy 36

iv

LIST OF FIGURES

Figure 1 Structural Kinetic Morphological and Fluorescence Characterization

of Amyloid Fibrils 5

Figure 2 Rigid Fibrils Lag Period Determination 18

Figure 3 Hen Egg White Lysozyme Native Structure 28

Figure 4 Hen Egg White Lysozyme Secondary and Tertiary Structure at pH 2

and pH 7 29

Figure 5 Structural Homology of Hen Egg White and Human Lysozyme 31

Figure 6 Static Light Scattering Dynamic Light Scattering and Atomic Forces

Microscopy Signatures of Rigid Fibrils Growth 33


Microscopy Signatures of OligomersCurvilinear Fibril Growth 35

Figure 8 Debye Ratio and Interaction Parameter for Hen Egg White Lysozyme

Monomers at pH 2 20ᵒC 37

Figure 9 Infrared Spectra of Late Stage Fibrils in Either Pathway Show

Distinct β-sheet Peaks 40

Figure 10 Amide I Spectra and Their Time Evolution for Lysozyme Undergoing

Oligomeric Fibril Growth 41

Figure 11 Thioflavin T Response to Hen Egg White Lysozyme Aggregation Kinetics 43

Figure 12 Hydrolysis Effects on Pathways and Thioflavin T Response During

Kinetics Measurements 45

Figure 13 Morphology and Kinetics of Rigid Fibrils Oligomers Curvilinear Fibrils

and Precipitates Formed by Hen Egg White Lysozyme 47

Figure 14 Kinetics Phase Diagram for Rigid Fibrils Oligomer and Curvilinear

Fibrils and Precipitate Formation of Hen Egg White Lysozyme 49

v

Figure 15 Oligomer vs Curvilinear Fibrils Formation Metastability of Oligomer

Phase Against Rigid Fibrils Growth and Amyloid Structure of Precipitates 52


Fibrils and Precipitate Experiment vs Theory 54

Figure 17 Supersaturation as a Driving Force for Pathway Switch 56

Figure 18 Growth Kinetics and Aggregate Morphologies Below and Above Critical

Oligomer Concentration 58

Figure 19 Schematics of Nucleated Conformational Conversion (NCC) vs Nucleated

Polymerization (NP) of Rigid Fibrils 61

Figure 20 Schematic of Anticipated Changes in Rigid Fibrils Lag Period Upon

Crossing of Critical Oligomer Concentration for Nucleated Polymerization

(NP) Nucleated Conformational Conversion (NCC) and Nucleated

Polymerization (NP) with aO Buffering 62

Figure 21 Lag Periods for Rigid Fibrils Nucleation Below and Above the Critical


Figure 22 Lag Periods for Rigid Fibrils Nucleation upon ―Seeding with Oligomers

and Curvilinear Fibrils Below the Critical Oligomer Concentration 65

vi

ABSTRACT

Deposition of protein fibers with a characteristic cross-β sheet structure is the molecular

marker associated with human disorders ranging from Alzheimers disease to type II diabetes and

spongiform encephalopathy Given the large number of non-disease related proteins and peptides

that have been shown to form amyloid fibrils in vitro it has been suggested that amyloid fibril

formation represents a generic protein phase transition In the last two decades it has become

clear that the same proteinpeptide can assemble into distinct morphologically and structurally

amyloid aggregates depending on the solution conditions Moreover recent studies have shown

that the early stage oligomeric amyloid assemblies are the main culprit in vivo We have

investigated the amyloid assemblies formed under denaturing conditions for Hen Egg White

Lysozyme (HewL) whose human homologue is directly implicated in hereditary non-neuropathic

systemic amyloidosis Our early investigations showed that HewL can aggregate via at least two

distinct assembly pathways depending on solution ionic strength at fixed pH temperature and

protein concentration By combining Dynamic Light Scattering (DLS) Static Light Scattering

(SLS) and Atomic Force Microscopy (AFM) we showed that at low ionic strength the pathway

is characterized by the nucleation and growth of long (several micron) rigid fibrils (RF) via

monomers assembly A second high ionic strength pathway is characterized by the rapid

assembly of monomers into globular oligomers that further polymerize into curvilinear fibrils

(aOCF) At NaCl concentrations above 400 mM aggregation resulted in precipitate formation

vii

Next we used Foureir Transform Infrared spectroscopy (FTIR) and an amyloid-specific dye

Thioflavin T (ThT) to show that both RF and (a)OCF are amyloidogenic species but they have

detectable structural differences Moreover we have determined that each assembly pathway has

unique SLS DLS FTIR and ThT response signatures that help determine the assembly type

prior to AFM imaging of aggregates

Taking advantage of the morphological structural and kinetic signatures for the two distinct

HewL amyloid aggregates I mapped out their amyloid aggregates phase diagram spanning over

two orders of magnitude in protein concentration and from 50 to 800 mM NaCl in ionic strength

This is the most complete phase diagram for amyloid aggregates of a given protein up to date

The phase diagram has three distinct regions delineated by sharp boundaries The RF- aOCF

was called Critical Oligomer Concentration and we commonly refer to ―above the COC as the

region were aOCF are kinetically favored In the region of low salthigh protein concentrations

RF were the only amyloid species to nucleate and grow As both salt and protein concentrations

increase aOCF become the kinetically favored species and RF nucleate and grow after several

days of incubation At high protein and high salt concentrations aOCF form very fast and

eventually lose solubility forming a precipitate (Ppt) Cross-seeding experiments showed that RF

is the thermodynamically stable aggregate phase while the OCF are the metastable species

Finally we used the phase diagram to design experiments that would allow us to reveal the RF

nucleation mechanism in presence of aOCF RF nucleation above the COC can undergo either

via internal restructuring of aOCF (NCC) or through a random coalescence of monomers into a

nucleus (NP) The experimental results obtained so far strongly indicate that RF nucleate via NP

mechanism both below and above the COC

1

I INTRODUCTION

I1 Amyloidosis as Health Care Challenge

Amyloidosis is the general term for a series of diseases characterized by extracellular

depositions of insoluble protein fibrils in tissues and organs 1ndash3

(table 1) The most familiar

amyloid diseases are Alzheimerlsquos disease (AD) Parkinson disease (PD) Huntington disease

(PD) systemic immunoglobulin light chain amyloidosis (AL) and the familial transthyretin

associated amyloidosis (TTR) Up to now nearly all amyloid diseases are incurable and present a

major healthcare and economic problem In 2015 alone 181 billion unpaid hours at an estimated

cost of 221 billion dollars have been spent by families and communities in the United States on

care giving for Alzheimer and other dementia patients4 Approximately 10 - 20 of people over

65 years has been estimated to have a mild cognitive impairment due to Alzheimer5 while

cardiac TTR amyloidosis occurs in 8 -16 in people over 80 years old6

The term ―amyloid is attributed to Virchow7 and it is directly translated as ―starch from Latin

Virchow noticed that the homogeneous portions of autopsy tissue stained with iodine and

sulfuric acid presented tinctorial characteristics (ie staining properties) similar to cellulose

hence he considered amyloid accumulations to have a carbohydrate molecular basis Amyloid

depositions in organs were described as homogeneous greasy pale yellow colloidal particles

and occasionally as white fibrillar outpourings237

Biochemical studies by Friedrich and

2

Table 1 Amyloid Diseases and Its Associated Polypeptides or Proteins

Disease Precursor protein Amyloid protein

Alzheimerlsquos disease Amyloid precursor protein Aβ peptides

Atrial amyloidosis Atrial natriuretic factor (ANF) Amyloid ANF

Spongiform encephalopathies Prion protein (PrPc) PrPsc

Primary systemic amyloidosis Immunoglobulin light and

heavy chains

AL and AH

Senile systemic amyloidosis Wild-type transthyretin ATTR

Haemodialysis-related

amyloidosis

β2-microglobulin Aβ2M

Hereditary nonneuropathic

systemic amyloidosis

Lysozyme ALys

Type II diabetes Pro-IAPP IAPP or ―amylin

Injection-localized amyloidosis Insulin AIns

Secondary systemic

amyloidosis

(Apo) serum amyloid A Serum amyloid A

Hereditary cerebral amyloid

angiopathy

Cystatin C ACys

Finnish hereditary systemic

amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I

Transthyretin variants ATTR

Familial amyloid

polyneuropathy II

Apolipoprotein A1 AApoA1

Ageing pituitary prolactinomas Prolactin APro

Familial amyloidosis Fibrinogen αA-chain AFib

Adapted with permission from Rambaran and Serpell Prion 2008 23 112-117 Table 12

3

Kekule in 1859 showed that amyloid fibrils were built from polypeptides however the term

―amyloid was not changed to reflect that8 and this term is exclusively used in the modern

literature

I2 Structural Hallmarks of ldquoClassicalrdquo Amyloid Fibrils

In the beginning of twentieth century Divry and Florkin showed that Congo Red a

cytoplasm and erythrocyte stain tightly binds to amyloid fibrils in tissue samples and exhibits an

intense birefringence with respect to the long axis of these deposits appearing apple green under

polarized light9ndash11

The birefringence indicated that these proteinaceous depositions were not

amorphous but rather had a well-defined underlining structure Further Thiovlavin T has been

identified to bind tightly to amyloid fibrils and it has been extensively used for studying growth

kinetics since this discovery in 195912ndash15

Electron microscopy done on tissue specimens showed ―the fine curved nature of the fibrils16

that were either disorderly distributed or in bundles depending on the organanimal Regardless

of animal or organ origin the fibrils had very similar widths of 05- 14 nm and length of 1-16

microm and their morphology was clearly distinct from any non-pathological protein fibril known at

that time1116

In 1968 the hallmark cross-β sheet structure of amyloid fibrils was resolved from

X-Ray diffraction of both human and animal amyloid tissue extracts In their article Eanes and

Glenner revealed the well-known rings at 475 Aring overlaying a diffuse halo at 43 Aring and a 98 Aring

ring present in non-oriented fibrils vs the ―cross-β X-Ray pattern from oriented fibrils17

4

In 1971 advancement in biochemical methods led to the identification of homogeneous

immunoglobulin light chains as first plasma proteins able to undergo amyloid fibril formation18

This was followed by the first in vitro growth of amyloid fibrils from κ and λ chains of Bence

Jones proteins at acidic pH and physiological temperatures mimicking renal tubes conditions319

The same year Benditt and Eriksen isolated a different protein that was also involved in amyloid

fibril formation and called it protein A20

In subsequent years several other proteins have been

reported to form amyloid fibrils in vitro and substantial work has focused on isolation and

biochemical identification of these proteins in human amyloid fibrils21ndash27

In 1998-99 in a series of four articles Kelly Dobson and Chiti established that amyloid fibril

formation is an inherent property of polypeptide chains They also showed that human proteins

under specific denaturing conditions undergo conformational changes of their tertiary and

secondary structure that promoted amyloid fibrils formation28ndash31

This hypothesis was further

supported by work from Aso et al who generated amyloid fibrils under denaturing conditions

from 25 non-disease related proteins32

Having established that amyloid fibril formation is an intrinsic property of the polypeptide

backbone scientists set out to find generic physical and biochemical parameters that would allow

one to better understand amyloidogenesis The starting point in this search was the amyloid

cross-β sheet since it is the common structural feature of all amyloid fibrils regardless of the

protein of origin3334

Using nuclear magnetic resonance (NMR) spectroscopy by 1998 it was

shown that Aβ fibrils have a parallel in register cross-β sheet35

In 2000 based on powder X-ray

diffraction of an amyloid forming yeast peptide David Eisenberg and his colleagues proposed a

model for amyloid fibrils as a dry (or dehydrated) in register parallel-stranded densely packed

5

β-sheet structure The model explained the stability of the cross-β sheet core as being mainly

entropically driven due to the large gain in entropy from water excluded from the rigid fibril

core with minor energy contributions from the hydrogen bonding of side chains among

(a)

(b)

(c)

(d)

(e)

(f)

Figure 1 Structural Kinetic Morphological and Fluorescence Characterization of

Amyloid Fibrils (a) characteristic kinetics curve for amyloid fibril nucleated polymerization

indicating the nucleation lag period growth (polymerization) period and the equilibrium

plateau43

(b) ThT fluorescence response upon binding to amyloid fibrils Notice the orders of

magnitude increase in fluorescence emission in presence of amyloid fibrils43

(c) Morphologies of

human lysozyme amyloid fibrils formed at pH5 10 TFE 68ᵒC and vigorous stirring (d)

characteristic X-Ray diffraction pattern of amyloid fibrils Notice the anisotropic reflections at

47 Å and 10 Å44 (e) cartoon schematics of amyloid fibrils β sheets and amyloid cross-β core

Notice the origin of the reflection at 47 Å and 10 Å observed in the diffraction pattern45 (f)

view of the dry steric zipper Notice the protruding residues and their steric accommodation

between the sheets backbones2

Figure 1 Structural Kinetic Morphological and Fluorescence Characterization of Amyloid

Fibrils (Continued on Next Page)

6

Fig IB1ab adapted with permission from Arosio et al Phys Chem Chem Phys 2015 17

7606mdash7618 Fig 3a and 6b respectively

Fig IB1df adapted with permission from Rambaran and Serpell Prion 2008 23 112-117

Figure 22

Fig IB1e adapted with permission from Smith et al Life 2014 4 887-902 Fig 1

themselves in the absence of water36

A few years later Eisenberg and his team put forward a

complete model of the cross-β spine of amyloid-like fibrils as a tightly self-complementing steric

zipper formed by the side chains bulging from two adjacent fibril sheets that helps to clamp the

sheets together3738

(fig 1) Subsequent investigations of short peptides derived from various

amyloid proteins lead to identification of eight different classes of zippers based on sheets

positioning and the compatibility39

of the side chains Using amyloid fibrils and a combination of

experimental techniques spanning over five orders of magnitude physical length Dobson and his

colleagues showed that the two dimensional β-sheets associate further into tertiary

protofilaments quaternary filaments and then fibrils40

The extensive work on amyloid fibrils

structure of Blacke Kirshner Eisenberg Dobson and other groups showed that the cross-β sheet

arrangement is a delicate balance between entropy (hydrophobic interactions

translationalrotational entropy of water molecules or peptide backbone side chains steric

contributions) and energy (hard core repulsion Coulomb interactions like charge-charge dipole-

charge or dipole-dipole with hydrogen bonding being a special and major contributor)33ndash42

I3 Amyloid Aggregation Pathways

Parallel to intensive studies done on generic long rigid fibrilslsquo amyloidogenesis evidence

was accumulated that proteins can concomitantly or sequentially explore various non-fibrillar

amyloid aggregate states under identical solution condition46ndash48

Well-defined early stage

7

aggregate populations have been identified both in vivo and in vitro48ndash51

and have been called

oligomers due to their small size It has been suggested that oligomers can further polymerize

into worm-likecurvilinear fibrils52ndash57

and nucleate the late stage rigid fibrils55ndash59

Initial in vivo

detections of early stage oligomeric species suggested that they are the most toxic amyloid

populations4647

and subsequently in vivo evidence that supported early in vitro findings was

gathered 63ndash66

Up to date there is still conflicting evidence regarding the toxicity of the early

stage amyloid aggregates66ndash68

A complete picture of amyloid fibrils formation has to incorporate early stages oligomeric

populations as well as end stage amyloid aggregates and be consistent with the structural data of

the cross-β sheet atomistic description It has become clear that amyloid aggregation is a

complex process and models describing this process have to account for both the generic

character of amyloid aggregation as well as the individual physical parameters of peptides and

protein 6970

Special attention has been dedicated to the fate of early stage oligomers and their

subsequent curvilinear fibrils since for the overwhelming majority of amyloid proteins they are

not the end stage amyloid product in vivo or in vitro 4864ndash6668

This latter conundrum highlights

the necessity of following the aggregation process in situ and identifying the relevant assembly

mechanisms at work at different time points

I4 Amyloid Aggregation Mechanisms

One of the most common approaches to study amyloid aggregation is to monitor its

kinetics in vitro for a wide spectrum of amyloid proteinspeptides The kinetic curves have been

8

extensively used to infer molecular details of the aggregation process and multiple molecular

mechanisms have been put forward based on the analysis of aggregation kinetics alone6971ndash7771

The study of protein polymerization kinetics in order to obtain details on molecular mechanisms

of supramolecular self-assembly goes back to work by Oosawa for actin polymerization79ndash81

His

model paved the way for nucleated polymerization aggregation mechanism for proteins and it

has remained the most dominant aggregation model until now In his works he introduced the

notion of critical concentration as the concentration threshold for aggregate nucleation Also

Oosawa group described the cooperative nature of actin fibrils polymerization and how the

nucleation lag period can be removed by seeding the sample with pre-formed fibrils In order to

get a meaningful physical model of actin polymerization process that would fit the empirical

data Oosawa and his colleagues had to make the distinctions between the substrate monomers

(G-actin) and the monomer state that is incorporated into polymersfibrils (F-actin) and assign

each actin type different energy and entropy contributions to the total energy of the system76ndash78

Discrepancies between actin and flagilin end stage polymerization experiments results and his

modellsquos predictions lead him to introduce two additional concepts fibrils depolymerization and

fragmentation and a description of the polymer size redistribution at the end of polymerization

when association and dissociation occur at comparable rates82

After Oosawalsquos description of

actin polymerization the model was modified to introduce a separate step describing the

nucleation event During their work on hemoglobin Eatonslsquo group described the

thermodynamically unfavorable nucleus8384

and assigned it a low rate of formation which lead to

a lag period associated with the nucleation event in the kinetics As a result of combining

Oosawa and his colleagues work and further developments from Eatonlsquos group the characteristic

9

sigmoidal shape of nucleated polymerization (NP) with three regions (lag period elongation

plateauequilibrium) became the protein aggregation kinetics signature85

It is essential to realize that the nucleated polymerization aggregation model was developed in

the context of fully reversible aggregation that did not require protein denaturation or chemical

or specific bonds formation during polymerization

Subsequent modifications of the nucleated polymerization model have been introduced based on

observations of different protein aggregation kinetic curves under denaturing conditions (ie

amyloid fibril aggregation) Scientists have tried to redefine the nucleus size the forward and

backward polymerization rates introduce different rates for each polymerization step different

rates for nucleation before and after the polymerization began or defined ―catalytic surfaces on

protein aggregates surfaces85ndash92

Additional modifications to the model were made to generalize

the nucleation event as an arbitrary step with the lowest rate and to allow (de)polymerization via

small oligomers not monomeric addition only93ndash9572ndash75

A crucial step for the study of protein aggregation mechanisms was the discovery of prion

proteins and their inherent infectivity by Pruisner2796

The model for prion aggregation was

based on Griffithlsquos mathematical model97

of Templated Assembly where the ―reactive

monomeric species P would be an isomer of the inactivenative monomer C The transformation

of C into P is thermodynamically unfavorable (they have very dissimilar energies) while the

formation of a P dimer from two P monomers is thermodynamically favorable After the

formation of the P dimer the C monomeric isomer is further transformed into the P isomer

through the interaction with the P dimer or larger subsequent aggregates In the end a mixing

between the nucleated polymerization and Griffithslsquo template assembly Nucleation-Dependent

10

Polymerization (NDP) was the most accepted model for prion aggregation and infectivity98

According to the model there is equilibrium between the C and P isomers of the same protein

The nucleus forms from the P form and has an assigned nucleation rate Once the nucleus forms

the equilibrium between the C and P tends to replenish the P population and hence serves as a

driving force for further conversion The further conversion of C into P is catalyzed by the

present P oligomers which are now in solution hence the growth is rapid98ndash100

In 2000 Lindquist and her lab proposed another model that combined yet again the Griffith and

Lansbury48101102

models and called it Nucleated Conformational Conversion (NCC) In this

model C the ―normal monomers form quickly the kinetically favored C oligomers (nuclei) that

undergo a slow internal conversion into the thermodynamically more stable P rigid fibril nuclei

hence the rigid fibrils nucleus formation is a two-step process The P fibril can further

polymerize from C oligomers or a C monomer The C oligomers or monomers undergo a rapid

internal restructuring upon interaction with the P polymer and the entire growth is fast58

So the

―infectious entity is the P oligomer (rigid fibril nucleus) and the polymerization occurs via the

converted C oligomers and C monomers While NCC has been shown to fit to various degree of

accuracy the kinetic curves for aggregation of several proteins58596162103

it still leaves open the

question of what are the molecular mechanism of the internal structural conversion of P

oligomers and how common the NCC protein aggregation scenario is

I5 Amyloid Species Phase Diagram(s) and Their Importance in the Field

Amyloid aggregation has been described in more detail in the context of statistical

thermodynamics as a region in protein phase diagrams104ndash108

or as a self-assembly process109110

11

This statistical mechanics framework allows to determine the role of short vs long range

interactions as well as the strength vs specificity of these interactions52104106107111ndash116

in

resolving the molecular mechanism(s) of amyloid aggregation under given solution conditions

Based on the nature and extent of the physical interactions between the protein monomers in

solution it is possible to build equilibrium phase diagrams that give a holistic picture of the type

and relationships of different amyloid aggregate phases One of the earliest attempts to build

experimentally aggregate phase diagrams was presented by Radfordlsquos group54

They used protein

concentration vs pH and protein concentration vs salt concentration to map out the type of

aggregates formed at different ionic strengths and β2-microglobulin concentrations in solution

At low ionic strengths the typical long straight amyloid fibrils would form via NP while at high

ionic strength solution conditions non-nucleation dependent (how do they know that if they only

monitored results after 3 weeks) worm like fibrils and oligomers would form The Radford team

monitored amyloid aggregation for three weeks so it is unclear if the species detected are the

end product of aggregation under the given solution conditions or they are the kinetically favored

amyloid state Also the protein salt and pH ranges are very limited and amorphous phases were

excluded from consideration

Several years later Goto and his team identified structureless aggregate phases for β2-

microglobulin they related to a glass phase and also defined a transition curve between the

amorphous and the fibrillar (crystal-like) aggregate states107

They showed that aggregation is

supersaturation driven and that the amorphous state is metastable with respect to the fibrillar

state Their work complemented the of the Radford team by identifying the amorphous phase

beyond the worm-like and long fibrils However their transition curve for the amorphous state

delimits amorphous aggregates as a generic state of proteins and not an amyloid state In

12

addition they do not show solubility curves of different amyloid species hence the phase

diagram they build is a protein phase diagram not an amyloid aggregates phase diagram The

mere identification of amyloid aggregates as a phase did not show the equilibrium or kinetic

relationship between the worm like fibrils and the long straight fibrils so this core question

remained unanswered

I6 Motivation and Goals for My Research

During my undergraduate work in Dr Muschollsquos laboratory I was fortunate to work with

the graduate student that identified and characterized the morphologies of HewL amyloid

aggregates under denaturing conditions52

We also showed that the net two-body interaction of

protein monomers in solution was a key physical parameter for HewL aggregation Working on

this project under her and Dr Muschollsquos guidance served as my main motivation to continue

working on revealing further physical parameters that affect the molecular mechanisms of

protein self-assembly processes

I have continued my work towards my set goal by working with our team on the next step

throughout my first year of graduate studies on characterizing the kinetics and structure of HewL

amyloid aggregates we previously identified117

The works of Radford and Goto presented in Section IE proved that amyloid aggregates phase

diagrams are powerful tools for understanding the kinetics and themodynamics of

amyloidogenesis The experimental evidence (including our own) that two-body electrostatic

interactions and initial concentration of protein monomers in solution play a crucial role in

13

dictating the aggregationlsquos outcome51107111-113

suggested that these are also two key parameters

controlling the aggregate phase space

Uniquely positioned to work with an amyloidogenic protein system where all three aggregates

states (long straight curvilinear and amorphous) I set out to map an entire phase diagram within

the protein concentration vs ionic strength phase space and to determine the relationship

between the amyloid aggregate phases Moreover the phase diagram would have provided us

with both a qualitative and quantitative understanding how various aggregation mechanisms

translate into the corresponding assembly kinetic curve signatures They would have also

allowed us to identify the driving forces and the molecular mechanism underlying amyloid

aggregation in our system

Once I mapped out the phase diagram for HewL amyloid aggregates we set out to use it in

answering one of the main conundrums in the amyloid aggregation field namely the nucleation

mechanism of rigid fibrils (the end stage aggregates) in presence of early oligomeric stage

amyloid aggregates

A holistic picture of both kinetics and thermodynamics of amyloid aggregation in vitro would

greatly contribute to understanding the clinical phenomenology of amyloid diseases and

contribute to designing molecular mechanisms to cure prevent or delay amyloid diseases

14

II MATERIALS AND METHODS

II1 Materials and Sample Preparation Protocols

In section 1 I describe in details the protocols for sample preparation and materials used

as well as curve fitting for kinetics data and extraction of lag periods for RF nucleation

II11 Fresh Hen Egg White Lysozyme Sample Preparation

Two times recrystallized dialyzed and lyophilized HewL purchased from Worthington

Biochemical Corporation (cat P00698) in buffer without additional NaCl at desired pH value

at double the desired final concentrations (25 mM KH2PO4 H3PO4 for pH2 (Sigma Aldrich

ACS reagent ge 99 CAS 7778-77-0 Fisher ACs grade CAS 7664-38-2) 100 mM C6H8O7

C6H5Na3O7 for pH5 (Fisher ACS CAS 5949-29-16132-04-3) and 20 mM HEPES (Fisher CAS

7365-45-9) or 100 mM NaH2PO4 Na2HPO4 for pH7 ACS reagent ge 99 ) The buffers are

pre-filtered through a 220 nm PES Corning bottle-top filter HewL samples were incubated at 42

ᵒC in a water bath for 2 minutes in order to dissolve any mesoscopic aggregates formed during

the lyophilization process After the water bath the samples were filtered through a 200 nm

Fischerbrand nylon syringe filter (cat 09-719C) and a 50 nm Thermo Scientific cellulose acetate

syringe filter (cat 03-376-230) The protein concentration was measured using absorption at

15

2801 nm with a ε280 = 264 ml(mgcm)

-1 and adjusted if needed through dilutions The HewL

samples were then gently mixed 11 with a buffer solution at double the desired final NaCl

(Fisher CAS 7647-14-5) concentrations

II12 Thioflavin T Stock Preparation

ThT stock was prepared by dissolving ThT powder (Anaspec Inc cat 88306) into DI

water from Millipore reverse osmosis system and the solution was filtered through the above

mentioned 220 nm and 50 nm pore size syringe filters The actual concentration was measured at

412120

nm using ε = 32000 M-1

cm-1

using UV Spectrometer (DeNovix DS-11 FX+) ThT stock

solution was stored at 4ᵒC for less than 30 days

II13 Rigid Fibrils and Curvilinear Fibrils Aggregates for Isolation

After the preparation of the HewL desired stock solution the final sample were obtained

by diluting the stock using the same salt buffer filtered through the 220 nm and 50 nm syringe

filters (see above stock preparation protocol) If multiple protein solutions concentrations were

needed usually a series dilution was used The sample were put in test 15 ml centrifuge tubes

(Thermo Scientific cat 339650) at 52 ᵒC in dry bath (Labnet AccuBlock Digital Dry bath) If

occasional monitoring with DLSSLS or ThT monitoring of aggregation was needed the sample

was incubated in a glass or quartz cuvette with stopper (see below DLSSLS measurements

protocol)

16

II14 Rigid Fibrils Isolation Protocol

RFs prepared under at the same solution as the solution they would be used as either seed

or initial RFs sample were isolated through a series of centrifugations The initial sample was

placed in a 2 ml centrifuge tube and spun down for 20-24 hours at 17700xg in centrifuge (Fisher

Scientific AccuSpin 1R) at 4 ᵒC The formed pellet would be re-dissolved in buffer and spun for

another 20-24 hours This cycle would be repeated until the concentration of the supernatant

after the centrifugation reached less than 1 of the pellet concentration The supernatant was

also checked with Dynamic Light Scattering (see below) to detect if there were any RFs

contaminants left in the supernatant After the last spin the sample was spun down at 5000xg for

30 minutes to precipitate any large or insoluble RFs clumps induced by centrifugation cycles

The RFs morphologies and pad presence of any residual clumps were checked with

Transmission Electron Microscopy (TEM see methods below) In order to check that the

centrifugation protocol was efficient at removing monomers after the last spin I dialyzed the

RFs at room temperature for 3 days and 6 changes of buffer and compared amyloid motif

identifying dye Thioflavin T fluorescence (see methods below) of the same nominal RFs

concentrations from dialyzed and non-dialyzed RFs samples The ThT fluorescence at 25ᵒC from

both dialyzed and non-dialyzed RFs samples were the same within experimental error indicating

that there were no significant differences between the sampleslsquo RFs concentrations

Isolating aggregates using cut-off filter was similar 400 mL of aggregated sample was put in a

50 kDa cut-off filter (Amicon Ultra cat UFC505024 ) and spun down at 15000xg for 15-30

minutes After the first spin 350 mL of the same (or exchange buffer if fibrils were to be used as

seeds at a different buffer salt concentration) was added and the sample was spun down at the

17

same centrifuge settings The last step was repeated 5 times At the end fibrils were recuperated

by inverting the filter in a new centrifuge tube and spinning it at 3000xg for 5 minutes and

dissolved in the buffer intended for further use

II15 Curvilinear Fibril Decay Experiments

We generated curvilinear fibril seeds and isolated them from the residual lysozyme

monomers via filtration using 100 kDa centrifuge cutoff filters The thermodynamic stability of

these seeds was tested by adding them to a series of solutions at fixed protein concentration (14

mM and 0692 mM) However a range of salt concentrations (0minus300 mM NaCl) was chosen to

cross the boundary for rigid filament-to-oligomer formation using 25 mM increments in NaCl

concentration For low protein concentration NaCl concentration was fixed at 350 mM and

HewL concentrations varied from 05 mM down to 0035 mM Total protein concentration was

taken as the sum of both monomer and seed concentration By using this approach the solution

conditions crossed the transition boundary perpendicular to its local slope and the experimental

accuracy improved

II16 Determination of Rigid Fibrils Nucleation Lag Periods

Protein solutions at concentrations ranging from 003 mgml (16 microM below the COC) to

5mgml (700 microM above the COC) were incubated in the presence of either 450 or 500 mM

NaCl Protein concentrations were more closely spaced near and below the COC for a given salt

concentration and more widely spaced above the COC The kinetics of three wells of identical

18

solution composition were typically averaged while the statistical variance was reported for

curve fitting and RF nucleation estimation times from each individual curve For quantification

of RF lag periods below the COC we defined the lag phase as the point at which the amplitude

of the ThT signal increases beyond a fixed threshold above the initial baseline Using semi-

logarithmically scaled ThT intensities significantly improves the sensitivity to detect the onset of

discernible RF growth from seeds nucleated up to that point Above the COC the initial growth

phase was fitted with a saturating (double)exponential over the range corresponding to aOCFs

growth (including the plateau until the second up rise in the kinetic curve) The fitted curve was

subtracted from the total curve and the resulting difference curve was assigned to correspond to

the parallel RF growth The lag period of RF nucleation was estimated using the same method as

for RF growth below the COC

(a)

Figure 2 Rigid Fibrils Lag Period Determination (Continued on Next Page)

19

(b)

Figure 2 Rigid Fibrils Lag Period Determination (a) For determining the lag period for RF

below the COC the lag period is fitted with a line and then a fix ThT net increase above this

baseline is taken as the lag period (b) For determining the lag period of RF above the COC the

curve is first fitted with a saturating exponential then the fitted curve is subtracted from the

experimental curve The difference curve is considered the curve corresponding to the RF growth

under these conditions The lag period is determined in the same way as for RF below the COC

II2 Methods and Measurements Parameters

In section 2 I describe in details the protocols for sample measurement parameters and

instruments used to obtain kinetic structural and morphological information on aggregation

behavior of hewL samples A detailed description of theoretical principles of the methods I used

in my PhD work are given in part B of the appendix

20

II21 Static and Dynamic Light Scattering Kinetics Measurements

Samples were incubated at 52 ᵒC in a 10 mm Starna glass cuvette topped with a PTFE

stopper in a SLSDLS instrument from Zetasizer Nano S Malvern Instruments Ltd The

instrument is equipped with a 3 mW He-Ne laser with 633 nm wavelength and measuring back

scattering at θ = 173ᵒ and it has a built in Peltier temperature control unit with a nominal

accuracy of plusmn 01 ᵒC Samples were measure at intervals ranging from 15 minutes to 30 minutes

and each data point was the average result of a 3 minute interval signal acquisition Sample

particle size distributions and their relative percent areas were obtained using the built in Contin

deconvolution analytical package Correlation functions were routinely checked to assure good

quality data acquisition Data was analyzed and plotted using Wavemetrics scientific graphing

data analysis image processing and programming software Igor Pro 5 All initial protein

solutions were prepared as indicated in Material section unless otherwise specified

II22 Thioflavin T Fluorescence Monitored Hen Egg White Lysozyme Amyloid Fibrils

Formation Kinetics

The bulk of the ThT fluorescence kinetics measurements were performed using a

SpectraMax M5 grating-based fluorescence plate reader (Molecular Devices) equipped with a

temperature-controlled sample chamber (Xdeg above ambient to 60deg C) ThT fluorescence was

excited at 440 nm and emission collected at 488 nm in presence of a 455 nm long pass filter to

suppress any scattered excitation light leaking into the emission channel 200-325 μL aliquots

were placed in a 96-well glass bottom plate (Cellvis P96-15H-N Fisher cat NC0536760) and

a ThT stock solution was added which brought its final concentration to 10-30 μM All

21

measurements were performed at 52 degC with the plate being shaken for 3-5 seconds every 10-20

minutes The plate was covered with either aluminum sealing tape (Corning Fisher

cat07200684) or transparent silicone matt (Thermo Scientific cat 03252163) Kinetic curves

were plotted using Igor Pro 5 (Wavemetrics) scientific graphing data analysis image processing

and programming software

Alternatively ThT fluorescence measurements were performed using a FluoroMax-4

spectrofluorometer (Horiba Jobin Yvon Edison NJ) equipped with a built-in Peltier temperature

control unit ThT fluorescence was excited at 445 nm and emission spectra were measured

between 465 nm and 565600 nm For each ThT fluorescence measurement small sample

aliquots were removed from the HewL growth solution and were mixed with the ThT stock

solution In order to minimize potential changes to the particle size distribution arising from

dilution during ThT measurements only 33 μl of the 212 μM ThT stock solution was added

directly to 667 μl of the undiluted lysozyme aliquots resulting in a final ThT concentration of

10 μM ThTHewL mixtures were transferred into small volume quartz cuvettes and allowed to

equilibrate for 5 min before recording ThT fluorescence emission For each fluorescence

measurement at least two separate ThTHewL solutions were prepared and their fluorescence

analyzed Offline ThT fluorescence measurements were taken at regular intervals (typ 2-6

timesday) while light scattering was monitored in situ as described above Correlated DLSSLS

and ThT measurements ceased when light scattering indicated the onset of sample gelation All

ThT measurements were taken at room temperature Using DLS we confirmed that the addition

of ThT the minimal dilution of the samples for ThT measurements or the lower temperature

used during fluorescence measurements did not noticeably alter the size distribution of HewL

22

aggregates35

All initial protein and ThT stock solutions were prepared as indicated in Material

section unless otherwise specified

II23 Fourier Transform Infrared Spectroscopy Measurement

Attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) was

performed on a Bruker Optik Vertex 70 (Ettlingen Germany) spectrometer with a mid-infrared

source and pyroelectric DLATGS (deuterated L-alanine doped triglycene sulfate) detector

Measurements were taken by placing 25minus30 μL of protein solution at 14 mM concentration on

the thermostated silicon crystal of a BioATRcell II (Harrick Scientific Products Inc

Pleasantville NY) accessory FTIR spectra were acquired between 1000 cm-1

and 4000 cm-1

All

spectra were taken at 24 degC with an aperture setting of 8 mm and a scanner velocity of 10 kHz

Typically 500-1000 scans at 4 cmminus1

resolution were recorded and three such runs were averaged

prior to data analysis Background spectra of the buffer solution without protein were recorded

over 200 - 1000 scans at 2 - 4 cmminus1

resolution and subtracted from the sample spectra Only data

that had a smooth flat baseline in the 1750 - 2000 cm-1

region were chosen as suitable for further

analysis Typically data analysis was performed on the average spectrum of three individual

runs The Amide I and Amide II bands (1500ndash1700 cmminus1) were deconvoluted together Peak

positions in the Amide I band of the spectra were identified using the Fourier Self-deconvolution

(FSD) (bandwidth 6 cmminus1 enhancement factor 24) and second derivative (13 smoothing points)

algorithms within the OPUS software analysis package (version 65 Bruker Optik) After a

horizontal baseline correction Gaussian curves were fitted from 1550 cmminus1

to 1700 cmminus1

The

overlap of the Amide I and Amide II band was best estimated by fitting the entire Amide I band

23

together with the high-frequency portion of the Amide II band Peak positions were fixed to the

values identified by FSD and second derivative algorithms while the intensity and widths of the

Gaussian curves were optimized using the Levenberg-Marquardt Algorithm To avoid

unreasonable broadening of Gaussian peaks observed for some spectra peak widths were

restricted to values near those obtained with native lysozyme For comparing spectra from

different fibril samples baseline corrected spectra were normalized to the integral over their

Amide I bands Difference spectra were obtained by subtracting the normalized reference

spectrum of monomeric lysozyme from the spectra of the aggregated samples These spectra

were renormalized by dividing each by the values of their respective β- sheet peaks

For measurements of the temporal evolution of the FTIR difference spectra sample aliquots were

removed at different times (4 h 1 day 4 days 7 days 11 days) and the initial monomer FTIR

spectrum at 25 C was used for subtraction121

All initial protein and ThT stock solutions were

prepared as indicated in Material section unless otherwise specified

Table 2 Amide I Band Assignment to Protein Secondary Structure Motifs Empirical vs

Theoretical Values Notice the higher discrepancy between the empirical and theoretical values

for α-helix This discrepancy may arise from the experimental difficulty to have reliable data in

that region of Amide I band due to the strong absorbance of water in the same exact region

Frequency (cm-1

) Secondary Structure

Associated

Theoretical Value

1620 - 1635 β-sheets 1627 (anti-parallel weak)124125

1636 - 1643 β-sheets 1640 (parallel)125

1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127

1689 - 1698 β-sheets 1690(anti-parallel

strong)128129

24

Band assignment of native of native HewL state was based on previous experimental122123

and

theoretical work which is summarized in table 2 above

The amyloid-β sheet band of FTIR spectrum has been assigned based on previous experimental

data from various groups that I summarized in table 3 below

Table 3 Comparison of Native vs Amyloid Fibrils Core β-sheets Notice that amyloid fibrils

core β-sheets emerge at lower frequencies than the native β-sheets for β2-microglobulin and

human lysozyme and appear de novo for Aβ42 and α-synuclein also at low wavenumbers

ProteinPeptide Aggregate Species Low Wavenumbers

β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)

Aβ42 Monomer (pH 74) - -

Oligomer (pH 74) 1626130

1695130

Fibrils (pH 74) 1620130

-

α-synuclein Monomers (pH 74) - -

Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-

β2 microglobulin Monomers (pH 75) 1633133

-

Oligomers (pH 21) 1618133

1684133

Fibrils (pH 21) 1620133

-

Human Lysozyme Monomers (pH 3) 1632134

-


1688134

Fibrils (pH7) 1626135

1693135

Fibrils (pH 2) 1622135136

-

25

II24 Atomic Forces Microscopy Imaging

AFM images were obtained in air with a MFP-3D atomic-force microscope (Asylum

Research Santa Barbara CA) using NSC36NoAl (Mikromasch San Jose CA) or PFP-FMR-50

(Nanosensor Neuchatel Switzerland) silicon tips with nominal tip radii of 10 and 7 nm

respectively The cantilever had a typical spring constant and resonance frequency of 2 nNnm

and 70 kHz respectively It was driven at 60minus70 kHz in alternating current mode and at a scan

rate of 05 Hz and images were acquired at 512 times 512 pixel resolution Raw image data were

corrected for image bow and slope using Wavemetriclsquos IgorPro 5 data analysis package For

aggregates 75 μL of sample solutions was diluted 20- to 200-fold into the same saltbuffer

combination used during growth deposited onto freshly cleaved mica for 3minus5 min rinsed with

deionized water and dried with dry nitrogen HewL monomers were not diluted and were

deposited on freshly cleaved mica for 30 minutes before rinsing with deionized water and dried

Amplitude phase and height images were collected for the same sample area False-color

heights were subsequently superimposed over either amplitude or phase images off-line using

Adobe Photoshop software

II25 Circular Dischroism Spectroscopy Measurements

Circular dichroism (CD) measurements were performed on an AVIV Model 215 (Aviv

Biomedical Lakewood NJ) or a Jasco J-815 (Jasco Inc Easton MD) CD spectrometer Spectra

of fresh HewL or amyloid aggregates were collected at 1 mgmL (Aviv) or 02 mgmL (Jasco)

and 25degC at pH2 or pH7 using a high quality 1 mm path length quartz cuvette Wavelength

scans were acquired between 190 and 260 nm in 1 nm increments For measurements with AVIV

26

spectrometer three scans with 10 seconds acquisition times each were averaged When using

Jasco 815 spectrometer three scans at a rate of 20 nmminute were averaged for each buffer and

sample In all cases buffer spectra were subtracted from the corresponding sample spectra

Temperature scans were performed between 20 degC and 90 degC in 5degC

II26 Gel Electrophoresis Experiments

HewL samples were analyzed using sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS PAGE) using 10ndash20 gradient Tris-tricine 12 lane gels (Criterion Bio-

Rad) and a sodium dodecyl sulfate (SDS) running buffer without glycine An aliquot of 20 μg

was mixed with Laemmli sample buffer (Bio-Rad) with or without reducing agent (β-mercapto-

ethanol) Samples were brought to 90ᵒC for 5 min loaded onto precast gels and stained with

BioSafe Coomassie (Bio-Rad) per manufacturerlsquos instructions For assessing the extent and time

course of lysozyme hydrolysis we incubated 1 mgml lysozyme solutions at pH 20 and 50 C for

0 to 120 h under either oligomer-free filamentous (50 mM NaCl) or oligomeric (175 mM) fibril

growth conditions The distribution of lysozyme fragments in solution and the extent of overall

hydrolysis were determined using both non-reducing and reducing SDS gel electrophoresis

27

III HEN EGG WHITE LYSOZYME

III1 Hen Egg White Lysozyme Biophysical Characteristics

Human lysozyme was first discovered by Fleming in 1922 in the nasal secretions of a

patient suffering from acute coryza and it drew his attention by exhibiting certain bacteriolytic

properties137

Soon it was discovered that its hen egg white homologue (HewL) is easily

accessible and researchers started to focus on HewL as a main research target in enzymology138

Its complete primary sequence was determined in 1963139

while its three-dimensional structure

was solved in 1965 by Blake and his team 106

This made HewL the first enzyme to have its

structure resolved by X ray diffraction107

The same team also investigated the activity of

HewL124

and in 1969 the catalytic mechanism of the enzyme was proposed143

HewL is a 129

amino acid protein with a molecular weight of 143 kDa and an isoelectric point of about 113

It has 6 tryptophans which allow accurate determinations of its solution concentrations using

UV absorption at 280 nm49122

one tyrosine and 2 phenylalanines which allow studies using

circular dichroism spectroscopy and eight cysteines forming 4 disulfide bonds conferring an

extraordinary stability to the molecule52145fig 2

HewLlsquos 3D structure is comprised of two main

sections with one section containing extended portions of alpha helices while the other section

contains mainly the disordered and beta sheet portions of the protein Based on its

crystallographic structure its secondary structure is 41 alpha-helical with 7 helices spanning

28

53 residues has 10 beta sheet content incorporating 14 residues while the rest is comprised of

disordered portions beta turns or remains unassigned145

(fig 3 )

(a)

(b)

Figure 3 Hen Egg White Lysozyme Native Structure (a) 3D ribbon diagram structure given

by X-Ray crystallography data (b) amino acid composition and associated secondary structure145

Figure adapted with permission from Protein Data Bank open source [PDB code 5L9J]

29

It has been shown that a combination of low resolution structural techniques (CD FTIR and

Raman Spectroscopy127128

) can yield reliable data on the secondary structure content of HewL

This allowed extensive studies of HewL structural changes induced by solution conditions (pH

temperature pressure ionic strength) or chemical modifications122146ndash148

(a)

(b)

(c)

Figure 4 Hen Egg White Lysozyme Secondary and Tertiary Structure at pH 2 and pH 7 (a) Denaturation Curve of 015 mgml HEWL pH2 (black) 50 mM NaCl measured using CD

and 5 microgml HEWL pH7 (red) no salt measured using Trp fluorescence (b) secondary structure

of HEWL at pH 2 (black) and pH 7 (red) measured with CD (c) FTIR spectra of fresh

monomers at pH 2 (black) and pH 7 (red)

III2 Hen Egg White Lysozyme Link to Familial Amyloidosis

The ability of human lysozyme to undergo amyloid aggregation was described by Pepys

in 1993 when it was detected that point mutations of the wild type human lysozyme (threonine to

isoleucine at position 56 (Thr56Ile) or histidine to aspartic acid at position 67 (His67Asp)) were

associated with familial non-neuropathic amyloidosis149

The first clinical study of this

amyloidosis was performed by Gillmore et al in 1999 showing that kidney failure from amyloid

depositions was the main pathological feature Liver and gastrointestinal system were affected to

30

a less degree and in sharp contrast to other non-neuropathic amyloidosis the heart was not

affected Patients are expected to live an average of 20 years after diagnosis150

Recently a new

type of mutation from arginine to tryptophan in position 82 has been discovered This

amyloidosis affects primarily the gastrointestinal system151

Today there are 8 points mutations

in human lysozyme known to induce amyloidosis (including the ones mentioned above)

Ile56Thr Asp67His Phe57Ile Trp82Arg Trp64Arg Thr70Asn Asp68Gly Trp112Arg151

Studies on structural stability of 4 out of 8 mutants (Ile56Thr Asp67His Phe57Ile Trp64Arg)

disease-related variants showed that they are characterized by the reduced thermal stability

which potentially renders them aggregation prone137138

Comparable thermal destabilization of

wild type lysozyme under denaturing conditions such as low pH and elevated temperatures

similarly induce amyloid formation in vitro It is interesting to notice that 7 out of these 8 disease

related point mutations affect the portion of lysozyme that has been shown to become part of the

amyloid core154

while the mutation at position 112 introduces steric stress by placing two

tryptophans next to each other

After Krebs et al showed that full length unreduced HewL can form amyloid fibrils under

denaturing conditions155

HewL became a valuable model for studying amyloid fibril formation

in vitro HewL has a 60 homology with the primary sequence of the human lysozyme156

but its

secondary and tertiary structure is essentially identical to that of human lysozyme (fig 5)

Multiple research groups have characterized the amyloid species formed by HewL under various

denaturing solution conditions As for native HewL low and high resolution techniques such as

X-ray diffraction circular dichroism spectroscopy (CD) Fourier Transform Infrared

Spectroscopy (FTIR) as well as ThT and Congo Red responses have yielded structural

31

information about HewL amyloid fibrils as well as for various oligomeric and curvilinear

intermediates34131139140142ndash144

(a)

(b)

Figure 5 Structural Homology of Hen Egg White and Human Lysozyme (a) HewL (green)

and Human Lysozyme (grey) structures superimposed using PyMol (b) the 57-107 amino acid

sequence of HewL (red) that has been shown to become a part of amyloid fibril core upon

aggregation154

Region was highlighted using PyMol Source files for structural information were

taken from Protein Data Bank open source [3FE0 5L9J]

32

IV RESULTS

IV1 Hen Egg White Lysozymersquos Two Assembly Pathways Kinetics Morphologies and

Spectral Properties

Our lab has carefully characterized the conditions under which various amyloid aggregate

species of HewL form in in vitro growth conditions Using SLSDLS (static light

scatteringdynamic light scattering) and AFM we determined that for fixed protein concentration

(14 mM) and fixed pH and temperature (pH 2 and 50 HewL can undergo amyloid fibril

formation via two different pathways1

At low ionic strength (0 -150 mM NaCl) aggregation followed the Nucleated Polymerization

(NP) kinetics as seen by SLS Under these conditions nucleation of the fibril seeds requires an

extended lag period of many hours to days Following nucleation the polymerization

(elongation) period is characterized by relatively modest increase in SLS We did not observe an

equilibrium plateau with SLS since the samples at our high protein concentrations formed clear

gels after growing for several days (fig 6a) This pathway also has a unique DLS signature

There is an extended lag period of several days when DLS only detects the monomer having a

hydrodynamic radius of 19 04 nm (fig 6b table 4) Immediately after the nucleation event

which is characterized by large fluctuations in SLS intensity and emergence of long tails in the

correlation function one observes an almost simultaneous appearance of two separate aggregate

33

peaks having radii around 30 nm and 300 nm respectively (fig 6b) This aggregation kinetics

were correlated with AFM images of samples taken at different time points along the aggregation

process Prior to nucleation no aggregate species were detected (fig 6c table 4) Shortly after

nucleation straight unbranched fibrils several micrometers long are observed with cross-

sections close to those of monomers This suggests that fibrils polymerize by monomer addition

(fig 6d table 4) At the late stages of aggregation fibrils underwent lateral assembly into larger

twisted fibrils We refer to this pathway as the oligomer-free pathway and the aggregates formed

as rigid fibrils (RF)

(a)

(b)

Figure 6 Static Light Scattering Dynamic Light Scattering and Atomic Forces Microscopy

Signatures of Rigid Fibrils Growth (Continued on Next Page)

34

(c)

(d)


Signatures of Rigid Fibrils Growth (a) SLS signature RF growth at 20 mgml HewL 50 mM

NaCl pH 2 52ᵒC Notice the long lag period ended by a sudden upswing indicating the RF

nucleation event (b) particle sizes given by DLS during aggregation under RF growth Notice the

small monomeric peak during the lag period followed by a bimodal aggregate peak distribution

characteristic for RF growth after nucleation (c) monomers observed during the RFs lag period

(d) vs rigid fibrils in the late stages of aggregation under RFs regime Scale bar indicates 50 nm

for (c) and 500 nm for (d)

At intermediate ionic strengths (150 ndash 350 mM NaCl) there is no signs of a nucleation event in

SLS and SLS intensity is undergoing a steady and accelerating increase (fig 7a) DLS showed

hydrodynamic radius of 2-24 nm near the monomeric peak that prior to nucleation underwent a

subtle increase by 05 to 1 nm In contrast to both SLS and ThT fluorescence DLS detects a

nucleation event resulting in the appearance of a single aggregate peak around 20 ndash 30 nm that

steadily increases in size to 50 ndash 60 nm (table 4 fig 7b) The samples gelled in a few days so

SLS and DLS monitoring was stopped AFM images taken during the aggregation process

showed small oligomers with volumes roughly corresponding to eight monomers after 3 hours

(fig 7c) and the nucleation event seen in DLS coincided with the polymerization of these

oligomers into curvilinear fibrils (fig 7d) We refer to this pathway as the oligomeric pathway

and the aggregates formed are amyloid oligomers and curvilinear fibrils (OCF)

35

(a)

(b)

(c)

(d)


Signatures of OligomersCurvilinear Fibril Growth (a) The SLS for 20 mgml HewL 250

mM at 52ᵒC starts to increase immediately upon reaching the denaturing temperature and no

feature indicates the polymerization of oligomers into CF (b) particle sizes given by DLS during

aggregation under OCF growth conditions in (a) Upon denaturation a broad distribution of the

monomeric peak is noticed hinting at the presence of small oligomers (bottom panel) At later

times a second peak corresponding to CF appears (c) small oligomers and occasional small

curvilinear fibril formed in the beginning of aggregation OCF growth (d) late stage curvilinear

fibril grown in the OCF regime presented in part (a) Scale bar indicates 500 nm

36

At high ionic strength (400 mM NaCl) disordered precipitate formed immediately upon

incubation as indicated by run-away SLS intensities and confirmed by AFM imaging We refer

to this region as precipitation (Ppt) and the aggregates formed are amorphous aggregates

Table 4 Summary of Rigid and OligomersCurvilinear Fibrils Morphologies Determined

by Atomic Forces Microscopy

Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)

Monomer (init) 19 plusmn 04 48 plusmn 08 31 plusmn 08

()

109 plusmn 24 226 plusmn 77

Monomer (pre-nucl) 21 plusmn 04 55 plusmn 09 40 plusmn 10

()


Rigid Filaments 24 plusmn 04 55 plusmn 06 104 plusmn 21

Rigid Mature Fibril 54 plusmn 03 72 plusmn 09 305 plusmn 43

Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)

Monomer 30 plusmn 02 38 plusmn 08 90 plusmn 20 227 plusmn 70

Oligomer 39 plusmn 01 95 plusmn 10 291 plusmn 32 184 plusmn 276

OligomericCurvilinear

Fibrils

39 plusmn 03 95 plusmn 17 291 plusmn 55

() || vs ζ to AFM scan direction

Table adapted with permission from Hill et al PLoS ONE 20116(4) e18171 table 152

The same pathways were identified when salts other than NaCl were used and for both divalent

anions and cations This confirmed that the ionic strength was a dominant control parameter of

the assembly pathway and non-specific charge screening by salt ions represented the dominant

mechanism driving the transition from rigid to oligomeric oligomer and curvilinear fibril

formation A counterintuitive finding about HewL aggregation was that the net two body

37

interaction of the monomers in solution under fibrillar growth conditions were repulsive which

is exactly opposite to conditions promoting protein crystallization159160

Given that at pH 2 the

net charge on HewL is +15e we put forward the hypothesis that at this pH and range of ionic

strengths the electrostatic charge-charge repulsion would be the force to dominate two-body

interactions In 2011 our lab established that the second virial coefficient obtained via SLS

measurements was positive (indicating indeed a net repulsive interaction) and decreased as salt

concentration was increased indicating the decrease in net repulsion At 400 mM NaCl where

amorphous precipitation occurred the second virial coefficient was close to zero (fig 8a)52

Furthermore the second virial coefficient together with the two-body interaction parameter

determined based on these experiments indicated that the balance between net repulsive and

attractive interactions had a clear correlation with the morphology and kinetics signatures of the

amyloid assemblies of HewL at pH2 (fig 8b)

(a)

(b)

Figure 8 Debye Ratio and Interaction Parameter for HewL Monomers at pH 2 20ᵒC (a) Notice the positive slope corresponding to the second virial coefficient which indicates a net

repulsive force (b) the interaction parameter calculated based on data from (a) show a clear

decrease of net repulsion as ionic strength of the solution increases and the morphologies transit

from rigid fibrils to oligomerscurvilinear fibrils and then to precipitates Table adapted with

permission from Hill et al PLoS ONE 201164 e18171 figure 5ab52

38

Overall these observations indicate that the aggregation pathway their kinetics and

corresponding aggregate morphologies were regulated by the effects of charge screening on both

the structure of the monomeric protein and intermolecular interactions between the monomers

IV2 Hen Egg White Lysozyme Aggregatesrsquo Structures and Their Time Evolution

In this section I describe how we synchronized our kinetics and topological

measurements of HewL aggregates growth with FTIR and thioflavin T dye to obtain structural

information on different types of aggregates

IV21 Probing Hen Egg White Lysozyme Aggregatesrsquo Structures and Their Time

Evolution Using Fourier Transform Infrared Spectroscopy

Beyond characterizing HewL aggregation pathways aggregate morphologies and

kinetics we also investigated their structural characteristics Structural analysis of aggregates

was performed using FTIR and ThT and correlated to the kinetics of fibril growth (DLSSLS) as

well as to changes in aggregate morphology (AFM)118

As a reference FTIR spectrum of

monomeric HewL under growth conditions were acquired and deconvoluted Results were

compared with reported literature values as control of both the experimental protocol and the

data analysis We opted for a conservative deconvolution using a minimal number of mainly α-

helix and β-sheet peaks to fit the spectrum The α-helix peak was located at 1655 cm-1

and the

combined β-sheet peak at low frequencies was found ~1620 cm-1

as determined from the second

derivative of the spectrum We found good agreement for the ratio of α-helix to β-sheet content

39

with that reported in the literature122

(fig 9a) FTIR spectra of both the end stage rigid fibrils

(RF) grown at low ionic strength as well as curvilinear fibrils (CF) grown at high ionic strength

exhibited distinct peaks in their Amide I bands around 1620 cm-1

158161

Peaks in this region of

the Amide I band are considered ―diagnostic of amyloid hydrogen bonding158

Despite the

similarities in the FTIR spectra of the RFs and OCF their amyloid peak positions showed a

subtle but consistent shift 1623 cm-1

for RF and 1619 cm-1

for OCF (fig 9bc fig 9a ) The

pattern of shifting the intermolecular β-sheet (~1620 cm-1

) band to lower wavenumbers

compared to native ones (1635 cm-1

) has been notices for several proteins as well as the rigid

fibrils band being at consistently higher wavenumbers that the curvilinearoligomers band (see

the table Appendix B32) In order to asses if the subtle differences in intermolecular β-sheet

position of RF and OCF are reproducible we took the difference of normalized spectra ie we

subtracted point by point the aggregatelsquos spectra from the initial monomeric spectrum As can

be seen in figure 10b difference spectra confirm the 1623 plusmn 1 cm-1

peak for RF and 1618 plusmn 1 cm-

1 peak for OCF These observations are consistent with results for human lysozyme fibrils and

oligomers grown under similar solution conditions (see table 3) Also from figures 9bc and 10b

it is easy to see that the intermolecular β-sheets forms at the expense of HewLlsquos α-helices for

amyloid aggregates in both pathways

The FTIR spectra of HewL aggregates confirm that at the end point of kinetics experiments the

aggregates contain amyloid-like -sheet peaks in the 1610-1630 cm-1

band but leaves open the

question when during the aggregation process this structure emerged Based on SLSDLS and

AFM data RFs form directly via Nucleated Polymerization ie their structure forms from the

40

(a)

Figure 9 Infrared Spectra of

Late Stage Fibrils in Either

Pathway Show Distinct β-sheet

Peaks Amide I spectrum for (a)

native lysozyme in buffer at pH

20 and for (b) oligomer-free or

(c) oligomeric fibrils analyzed

after incubation Black lines

represent the raw spectra while

gray lines indicate the gaussian

decomposition of the spectra into

α-helix β-turn and β-sheet

components as well as the

resulting overall fit to the raw

spectra The bar graphs to the

right of each spectrum represent

the relative amplitudes of the α-

helix and β-sheet peaks derived

from spectral In order to avoid

monomeric contributions to the

spectra monomers were separated

from the aggregates by

centrifugation through 50 kDa

cut-off filters Figure adapted

with permission from Foley et al

Journal of Chemical Physics

2013 139(12)121901 figure 2

(shared first authorship)

(b)

(c)

start In contrast the structural evolution of β-sheet content in oligomers and curvilinear fibrils is

not known Therefore we decided to monitor the development of the intermolecular β-sheets

41

content as function of incubation period Figure 10c shows that the amyloid-related β-sheets

motif can be detected in FTIR spectra as early as 4 hours after the incubation when DLS showed

no presence of CF yet

(a)

(b)

(c)


Oligomeric Fibril Growth (a) Amide I spectra of monomers at pH 2 (black curve) RFs grown

at 20 mgml (14 mM) HewL 50 mM NaCl pH2 52 separated through a 50 kDa cut-off filter

(red curve) and OCFs grown at 20 mgml HewL 200 mM NaCl pH 2 52 separated through

a 50 kDa cut-off filter (blue curve) (b) Corresponding FTIR difference spectra of the Amide I

region for oligomer-free fibrils (red curves) and for oligomeric protofibrils (blue curves) at pH

20 The two difference spectra for oligomer-free fibril growth are for samples either incubated

for multiple days (solid red curve) or derived from rapid growth (lt12 h) via seeding with pre-

formed fibrils (short-dashed red curve) Similarly oligomeric difference spectra are shown for

growth with 175 mM NaCl (solid blue curve) during rapid growth (lt3 h) in 325 mM NaCl

(short dashed blue curve) or for growth at pH 7 65 degC (blue dotted curve) (c) Temporal

evolution of the Amide I difference spectra for lysozyme undergoing fibril growth along the

oligomeric pathway Figure adapted with permission from Foley et al Journal of Chemical

Physics 2013 139(12)121901 figure 3 (shared first authorship)

IV22 Time Evolution of Aggregatesrsquo Structure Using Thioflavin T

To characterize further the structural evolution of amyloid aggregates in both pathways

we incubated solutions forming aggregates in each pathway extracted aliquots at different time

points and measured their ThT response ThT is a fluorescent dye that recognizes amyloid beta

sheet motifs in different amyloid fibrils as well as other naturally occurring non-disease related

42

protein fibers12ndash14

There is a remarkable difference in SLS vs ThT responses in each pathway

(fig 11) During RFs growth ThT undergoes a nearly 100 fold increase in fluorescence intensity

but its response to curvilinear fibrils is an order of magnitude lower and it shows virtually no

response to precipitates (fig 11) ThT response clearly follows the shape of the kinetic curves for

rigid fibrils and curvilinear fibrils growth respectively (fig 11 ab top) For rigid fibrils both

SLS and ThT showed no increase during the lag period and a sudden increase upon fibril

nucleation with the increase in ThT fluorescence being 40 fold higher initially (fig 11 a) This

behavior is also consistent with DLS measurements for aggregation behavior under RF solution

conditions where no discernable aggregated populations are detected during the lag period and

the nucleation event is sudden and characterized by the (almost) simultaneous emergence of a

double aggregates peak (fig 11a bottom) This pattern of correlated SLSThT fluorescence

behavior was noticed for several solution conditions for RF growth strongly indicating that this

is a general characterization of RF growth kinetics (fig 11a top bottom) For curvilinear fibrils

in contrast both SLS and ThT increase from the start which corresponds to oligomer formation

but no sudden changes in ThT fluorescence occurs as DLS indicates that the oligomers start

polymerizing into curvilinear fibrils (fig 11b) Again the pattern of correlated SLSThT

fluorescence behavior was noticed for several solution conditions for aOCF growth strongly

indicating that this is a general characterization of RF growth kinetics (fig 11b top bottom)

This serves as evidence that there is no significant restructuring of oligomers as they assemble

into curvilinear fibrils and polymerization did not result in any additional restructuring gain or

loss of amyloid motifs recognized by ThT Based on ThT and FTIR data we decided to call

oligomers (Os) as amyloid oligomers (aOs)

43

(a)

(b)

(c)

(d)

Figure 11 Thioflavin T Response to Hen Egg White Lysozyme Aggregation Kinetics (a)

(top) Relative changes in ThT fluorescence intensity () and light scattering intensity ( )

measured for a solution undergoing fibril growth under RFs growth conditions (6a bottom

sample) The sudden upswing in both ThT fluorescence and light scattering intensity coincides

with the nucleation of rigid fibrils detected by dynamic light scattering ) (bottom) Temporal

evolution of aggregates obtained from dynamic light scattering for a solution undergoing fibril

Figure 11 Thioflavin T Response to Hen Egg White Lysozyme Aggregation Kinetics

(Continued on Next Page)

44

growth under RFs growth conditions (50 mM NaCl) The plot shows a characteristic monomer

peak ( Rh = 19 04 nm) during the lag period until suddenly two additional aggregate peaks

emerge with peak radii near 300 nm and 30 nm respectively (b) (top) Relative changes in ThT

fluorescence intensity () and light scattering intensity () measured from the same sample For

CFs growth both ThT fluorescence and light scattering intensity show a continuous increase

with no sudden changes in behavior upon CFs polymerization seen in DLS (bottom) Temporal

evolution of the particle size distribution obtained from DLS for a solution undergoing OCFs

under monomeric OCFs conditions (175 mM NaCl) The slow increase of Rh of the monomer

peak indicates the formation of small populations of oligomers The aggregate peak at approx 30

nm emerging around 15 hrs indicates the formation of CFs polymerized from oligomers (c) Log-

log plot of the fractional changes in ThT fluorescence vs light scattering intensity for multiple

lysozyme samples incubated under conditions resulting in RFs growth (solid symbols) CFs

growth (open symbols) or high salt concentrations inducing amorphous precipitation (crosses)

The dashed lines represent linear fits through the data In all cases ThTSLS ratios display strong

and persistent linear correlations throughout the entire growth process (d) fractional changes in

ThT fluorescence with respect to light scattering intensity as a function of salt concentration

Notice that salt does not qualitatively affect the ratio Insert ThT fluorescence of a fresh 13 mM

HEWL sample at pH2 as a function of NaCl concentration Notice that over a 400 mM NaCl

span there is a double in ThT fluorescence most probably due to viscosity increase117

Figure

adapted with permission from Foley et al Journal of Chemical Physics 2013 139(12)121901

figure 568 (shared first authorship)

IV23 Investigation of Hydrolysis on Aggregate - Specific Thioflavin T Response

The observed differences in the structure of different amyloid species raise the question

whether those differences arise from different monomeric states being incorporated into RFs than

into OCFs Given that solution conditions (pH 2 and 52 ᵒC) favor hydrolysis147162

if hydrolysis

is salt dependent differences in OCF and RF structure could have roots in this dependence

However as can be seen from figure 12a hydrolysis does not depend on salt RFs and OCF

successfully incorporate the same monomeric states and structural differences are driven by

assembly mechanism rather than by monomeric state distributions Additionally we addressed

whether pathway-specific differences in ThT responses arose from the differences ingrowth

conditions or from binding to hydrolyzed HewL monomers Toward that end we measured with

ThT and FTIR HewL aggregates growth near physiological conditions (pH 7 elevated

45

temperature) DLS and AFM images of aggregates formed at pH 7 show that aggregation follows

the oligomeric assembly pathway (data not shown but can be seen in detail in Mulaj et al163

)

Upon comparison between figure 11b top and Figure 12b it is evident that ThTSLS ratio reveals

very similar patterns and no detectable restructuring of oligomers occurs as they assemble into

curvilinear fibrils This served as confirmation that structural evolution of aggregates formed

along oligomer-free and oligomeric pathways is independent of hydrolysis effects These results

reinforce the early obtained by FTIR where we showed that the intermolecular β-sheets is

present in oligomers and does not undergo any detectable transformations upon polymerization

into CF

(a)

(b)

Figure 12 Hydrolysis Effects on Pathways and Thioflavin T Response During Kinetics

Measurements (a) Reducing SDS PAGE gel of HEWL monomers incubated at pH 2 52 at

50 mM and 175 mM NaCl At each time step the right lane corresponds to growth at50 mM and

the left lane to 175 mM NaCl The extent of hydrolysis is independent of ionic strength (b) ThT

and light scattering ratios measured for fibril growth of lysozyme at physiological pH (pH 7 20

mM NaCl T= 65 ᵒC) Notice the striking resemblance of ThT and SLS at pH 7 (in absence of

hydrolysis) and pH 2 (in presence of hydrolysis) Figure adapted with permission from Foley et

al Journal of Chemical Physics 2013 139(12)121901 figure 48 (shared first authorship)

46

IV3 Kinetic Phase Diagram and Critical Oligomer Concentration (COC)

This section describes how I mapped the HewL amyloid aggregates phase diagram via

kinetic experiments in the HewL vs NaCL concentration phase space I have identified three

main regions where either RF aOCF or PPt were the kinecally favored aggregate The boundary

between RF and aOCF is sharp and it has been named Critical Oligomer Concentration (COC)

while PPt are just kinecally arrested aOCF Also via cross-seeding experiments we determined

that RF are the thermodynamically stable phase while aOCF are metastable long-lived

oligomeric species Further in collaboration with Dr Schmit from Univeristy of Kansas we

showed that a charged colloidal model captures the main parameters of aOCF formation as a

high order kinetics electrostatically driven phase transition

IV31 Mapping the Hen Egg White Lysozyme Amyloid Phase Diagram

The main motivation for the subsequent experiments was to determine whether there are

well defined ―transition boundaries delineating the formation of either rigid fibrils or oligomers

and curvilinear fibrils Specifically I wanted to explore whether charge effects (protein charge

and salt-mediated charge screening) would explain such transitions Utilizing the distinct

SLSDLS and ThT kinetic signatures of HewL amyloid aggregates grown in different pathways

I therefore mapped out the aggregation behavior of HewL solutions protein concentrations

covering a range of monomer concentrations from 6 μM to 14 mM and ionic strengths (50minus800

mM NaCl) while fixing fixed pH (= 2) and temperature (= 52 ᵒC) Figure 13 describes the main

criteria used to determine the assembly pathway from growth kinetics observed at a given protein

and salt concentration 138

SLSDLs and ThT kinetics were used to determine the pathway-

47

specific kinetics while AFM was used to confirm aggregate structure near the boundary were

kinetics becomes ambiguous

(a)

(b)

(c)

Figure 13 Morphology and Kinetics of Rigid Fibrils Oligomers Curvilinear Fibrils and

Precipitates Formed by Hen Egg White Lysozyme Summary of the distinct morphologies and

kinetics signatures associated with growth of (a) rigid fibrils (RF) (b) oligomers and curvilinear

fibril (OCF) and (c) precipitation (Ppt) of HewL under partially denaturing conditions (pH 2 T

= 52 degC) The first column displays atomic force microscopy images of RFs (50 mM NaCl) CFs


Precipitates Formed by Hen Egg White Lysozyme (Continued on Next Page)

48

200 mM NaCl) and Pps (400 mM NaCl) Black scale bar in all images represents 300 nm

Aggregate heights in all images are indicated by false-color with the corresponding scale given

below the bottom image The subsequent columns show representative thioflavin T (ThT)

responses static light scattering intensities (SLS) and particle size distributions autocorrelation

functions measurements with dynamic light scattering (DLS) RF growth displays a clear lag

period in all three signals RF nucleation causes the formation of two separate aggregate peaks in

DLS the sizes of which remain essentially stationary Oligomer formation and subsequent CF

nucleation and growth produce continuous increases in ThT and SLS CF nucleation only

generates one aggregate peak and does not yield a discernible discontinuity in ThT or SLS

responses Precipitation in turn induces rapidly saturating ThT and SLS signals with the

corresponding DLS autocorrelation functions exceeding conditions suitable for size analysis164

Figure adapted with permission from Miti et al Biomacromolecules 2015 16 (1) pp 326ndash335

figure 1

The resulting HEWL amyloid phase diagram is given in figure 14 Each point in the diagram

corresponds to the results from multiple kinetic measurements at these solution conditions Rigid

fibrils (RF) are the sole aggregated phase in the range of low salt or low protein concentrations

covered in our experiments As we increased either the ionic strength or protein concentration

amyloid growth abruptly switched to yield amyloid oligomers with their subsequent

polymerization into curvilinear fibrils (aOCF) We labeled this sharp boundary between the two

aggregated amyloid phases ―critical oligomer concentration or COC because of its resemblance

to the critical micelle concentration (CMC) in surfactant systems As either protein or salt

concentrations were pushed to higher values the solution formed amorphous precipitates (Ppt)

due to high aggregation driving force Similar to the determination of the COC we were able to

determine experimentally a boundary between the aOCF phase and the Ppt but given the fast

kinetics and the limitations of the methods used we must consider this boundary subject to larger

systematic errors than the COC

49

(a)

(b)

Figure 14 Kinetics Phase Diagram for Rigid Fibrils Oligomer and Curvilinear Fibrils and

Precipitate Formation of Hen Egg White Lysozyme (a) linear scale Kinetic Phase diagram of

amyloid aggregate species for hew-L undergoing amyloid growth as function of protein and

NaCl concentrations at pH 2 T = 52 degC Orange dots (bull) represent growth of rigid fibrils (RF)

blue dots (bull) indicate oligomer andor curvilinear fibril (aOCF) formation while dark blue dots

(bull) signify precipitation (Ppt) Aggregate species obtained at a given combination of protein and

salt concentration was typically determined from multiple kinetics measurements using ThT

SLS and DLS Dashed lines are guides to the eye highlighting the well-defined transitions

between the three distinct regions in this phase space for each of the three aggregate species (b)

the same data as in part (a) but on logarithmic scale Figure adapted with permission from Miti

et al Biomacromolecules 2015 16 (1) pp 326ndash335 figure 2

IV32 Amyloid Oligomer and Curvilinear Fibrils Share a Common Transition Boundary

There are three morphologically distinct aggregates that emerge past the COC amyloid

oligomer CF and Ppt Below the precipitation threshold the formation of CF is separated from

the onset of oligomer formation by a lag period This nucleation barrier implies that oligomer

concentrations need to exceed an as yet unknown solubility limit for CF We set out to establish

this solubility boundary by seeding solutions with preformed CF and then delineating the

conditions for the transition from growth to decay At high proteinlow salt concentrations

50

preformed CF were diluted into a series of solutions at fixed HewL concentration but decreasing

salt molarity (fig 15a) Conversely for low proteinhigh salt concentrations CF seeds were

diluted with different amounts of buffer solution at fixed salt concentration (data not shown)

Using DLS growth and decay rates of the CF peaks were monitored and extracted from

exponential or linear fits to the peak intensities versus time CF solubility was taken as the

extrapolated point where CF growth switched over to decay Within experimental error CF

solubility matched the COC and we therefore refer to this region by the single acronym aOCF

This also implies that oligomers under these conditions will always form CFs over time Despite

the polymerization of aO into CF we have found earlier that there is no detectable restructuring

These data combined suggests that aO polymerization would have no energetic barrier to

overcome

IV33 Amyloid Oligomers and Their Curvilinear Fibrils Are Thermodynamically

Metastable

The diagram indicated that amyloid aggregate species have well defined regions of phase

space where they are the kinetically favored state This however does not determine whether

these phases are thermodynamically stable or whether some are metastable In order to establish

this we seeded RFs grown below the COC into conditions above the COC If RF decay upon

crossing the COC then both RFs and aOCF represent the thermodynamically stable states in

their respective growth regimes If RF decay when seeded above the COC then RF represents

the thermodynamically stable state throughout the entire region Separated RFs grown under

solution conditions (see I14) where they are the kinetically favored state were cross seeded

into fresh monomeric solutions promoting aOCF growth (RF above the COC) As can be seen

51

from figure 15c and b RF continued to grow even above the COC This showed that RFs are the

stable state while OCF are a metastable aggregate phase I have also confirmed with AFM

imaging (data not shown) at a later time that the predominant aggregate species just after 24

hours is RF

IV34 Precipitation Boundary Structural Analysis via Infrared Spectroscopy

Since the phase diagram has a second boundary between the OCF and Ppt we wanted to

investigate whether this was a transition from an amyloid phase107

to an amorphous monomeric

aggregate phase or was an independent transition delineating yet another amyloid aggregate

phase of HewL FTIR was used to see if there was an underlying structure of Ppt If Ppt were a

random aggregation of native monomers we expected that there would be no discernable

differences in the FTIR spectrum of Ppt from that of HewL monomers To our surprise

precipitates formed at high saltprotein concentrations developed a pronounced β-sheet peak at

the same wavenumber as aOCF The weak peak near 1690 cmminus1

of OCF was less prominent for

precipitates and the peak near 1655 cmminus1

suggests that a residual fraction of α- helical structures

remains These differences probably arise from the difficulties of separating precipitates from

their monomeric background via cutoff filters which is less effective than for aOCF

Nevertheless the above IR spectra imply that the precipitates share structural characteristics of

oligomeric amyloids Their alignment with the aOCF peak in the Amide I band further implies

that the precipitates originated via rapid formation and subsequent precipitation of amyloid

oligomers and we refer to them from here on out as aO-Ppt The FTIR spectrum of Ppt indicate

that Ppt are ta kinetically trapped aOCF phase (fig 15d)

52

(a)

(b)

(c)

(d)

Figure 15 Oligomer vs Curvilinear Fibrils Formation Metastability of Oligomer Phase

Against RF Growth and Amyloid Structure of Precipitates (a) CF seeds grown at Clys = 20

mgmL and [NaCl] = 350 mM were subsequently re-suspended into solution with [NaCl]

increasing from 100 to 300 mM The transition from growth to decay of CFs occurred at the

same NaCl concentration as delineated by the COC These data indicate that there is no barrier

for oligomer formation and the lack of hysteresis so within experimental error the solubility of

oligomers is identical to that of CFs (bc) RF seeds grown at Clys = 20 mgmL hew-L and [NaCl]

= 50 mM were separated from monomers and seeded at 5 solution into monomer solutions

with Clys = 20 mgmL and NaCl between 100 and 250 mM (b) Changes in the particle size

distribution of RFs seeded into 20 mgmL hew-L monomers at [NaCl] = 250 mM s Both the

monomer peak (~ 2 nm) and the two aggregate peaks (short and long rigid RFs) are visible right

Figure 15 Oligomer vs Curvilinear Fibrils Formation Metastability of Oligomer Phase Against

RF Growth and Amyloid Structure of Precipitates (Continued on Next Page)

53

after seeding (0 hr) With increasing incubation period (10 hrs 20 hrs) the area under the RF

peak grows at the expense of the monomer peak with very large aggregates settling out near the

end (c) ThT fluorescence responses for RFs seeded into fresh lysozyme (16 mgmL) at NaCl

concentrations below (100 150 mM) and above (175 200 amp 250 mM) the COC In all cases

RFs continue to grow rapidly (d) Attenuated total reflectance FTIR spectra within the Amide I

band for amyloid RFs ( ) CFs () Ppt ( ) formed under the conditions used to determine the

phase diagram in Fig 2 Notice the peaks near 1620 cm-1

in all three aggregate spectra including

that for precipitates which are considered diagnostic of amyloid beta-sheets structures Figure

adapted with permission from Miti et al Biomacromolecules 2015 16 (1) pp 326ndash335 fig 3

IV35 Model of Colloidal Charge Repulsion Reproduces Critical Oligomer Concentration

Boundary

In order to model our solubility curves and gain a better understanding of the physical

parameters governing amyloid self-assembly we used a colloidal model that accounts for the

electrostatic energy cost of confining charged monomers within the volume of an amyloid

oligomer Implementation of the model assumptions derivations of analytical approximations

and numerical simulations were all performed by Dr Jeremy Schmit at Kansas State University

Within this model the COC is calculated as the free energy difference between the free energies

of the individual monomers vs that of the corresponding oligomer It accounts for the

electrostatic repulsive interactions among the monomers and a non-electrostatic and solution-

independent cohesive energy The rapid decrease in the COC with increasing salt concentration

arises from the increased charge screening of the net monomer charge The calculated solubility

curve fitted the experimental COC curve very well (fig 16b) In order to replicate the

precipitation boundary Dr Schmit had to assume further that oligomers had asymmetric

interactions with end-to-end interactions stronger than equatorial interactions This fact would

have remained unnoticed had we not tried to model the aOCFs to Ppt solubility boundary The

ability of a simple theoretical model to capture the complexities of the transition from oligomer-

free to oligomeric amyloid assembly is a strong indication that important aspect of amyloid self-

54

assembly are accessible to simplified models of colloidal self-assembly For further details on the

theoretical model please see Appendix A

(a)

(b)


Precipitate Experiment vs Theory Kinetics phase diagram of HewL amyloid aggregate

species as function of protein and NaCl concentrations at pH 2 T = 52 degC (a) Blue squares ()

and their error bars indicate the experimentally determined COC for HewL amyloid oligomers

dark blue squares () represent the onset of precipitation determined experimentally while the

solid blue and dark blue lines through the experimental oligomer phase boundaries are based on

theoretical predictions from the colloidal model (b) the same data as in part (c) of the graph but

on a logarithmic scale for a better visualization164

Figure adapted with permission from Miti et

al Biomacromolecules 2015 16 (1) pp 326ndash335 figure 4

IV4 Hen Egg White Lysozyme Rigid Fibrils Nucleation Mechanisms Above and

Below the Critical Oligomer Concentration

In this section I describe how I use the phase diagram for HewL amyloid aggregates to

answer one of the main questions in the field namely via which mechanism RF substitute early

stage aOCF there are currently two possible scenarios One scenario implies an internal

restructuring of aOCF into a RF nucleus while a second possibility suggests that aOCF are off-

pathway metasble aggregates that do not restructure into a RF nucleus and RF nucleate from

55

monomers undergoing classical nucleated polymerization We use the predictions on RF lag

period lengths in this two different scenarios and use our phase diagram to test this predictions

We found that RF undergo nucleated polymerization both in absence and presence of aOCF

Moreover there are stromg hints that aOCF actively inhibit RF nucleation

IV41 Amyloid Nucleation and Growth Kinetics Reflect Basic Aspects of the Amyloid

Phase Diagram

The phase diagram provides basic insights how RFs will emerge at the end stage of

amyloid assembly Based on the phase diagram the metastable phase of aOCFs is in equilibrium

monomer and would eventually decay back to monomers below their solubility COC and The

dimensionless super-saturation for aOCF formation is set by

= ( - COC)COC

where COC represents the aOCF solubility and represent the free monomer

concentration Meanwhile RFs can nucleate via NP at any point in the phase space above their

respective solubility including parts of the phase diagram where aOCF are the kinetically

favored state The thermodynamic driving force for RF nucleation and growth is the

corresponding supersaturation

= ( - SRF) SRF

where is the free monomer concentration and SRF the fibril solubility Our experimental

phase diagram provides an upper bound on SRF of a few micromolar (with SRF itself expected to

be noticeably smaller) However in contrast to aOCF the initial growth kinetics of RF is

56

limited by the high barrier for seed nucleation Using these concepts we can provide a basic

understanding why amyloid growth will proceed in distinctly different ways above and below the

COC

At solution condition above the COC the initial growth rate for aOCF is set by aOCF which

decreases to zero as monomers are converted into aOCF and Cmono reaches the COC While RFs

have a much lower solubility than aOCF their growth is retarded by the high nucleation barrier

for seed formation Therefore aOCF can be the dominant early-stage aggregate species and

even deplete monomers down to the COC before any noticeable levels of RF seeds can form (fig

17b) Eventually though RF will nucleated and begin elongating thereby depleting monomer

levels below the COC Therefore aOCF will start decaying and RFs will continue to grow until

all monomers tied up in the aOCF state are transferred to RF (fig 17b c)

(a)

(b)

(c)

Figure 17 Supersaturation as a Driving Force for Pathway Switch (a) Schematic

representation how aOCF are transformed into the RF state AFM image of aOCF and RF are

representations of commonly observed aggregate species in the aOCF and RF pathways Scale

with initial solution conditions above the COC As aOCF grow the monomeric substrate gets

Figure 17 Supersaturation as a Driving Force for Pathway Switch (Continued on Next Page)

57

depleted and the sample reaches the COC (blue dashed line) As RFs nucleate the monomeric

substrate gets further depleted toward SRF and the monomeric concentrations drops below the

COC (red dashed line) where aOCF can no longer grow or even be stable (c) The expected

kinetic signature g of a sample with initial conditions indicated in (b) Notice the rapid growth of

aOCF with an eventual slow down and plateau as monomer concentrations reach the COC A

second upswing is due to RFs nucleation and their subsequent growth

This logic is reflected in the growth kinetics as monitored with ThT for solutions below and

above the COC (fig 18a and b) Below the COC RF growth undergoes nucleated

polymerization As a result no change occurs to the ThT baseline during the lag period followed

by a sharp and explosive increase upon RF nucleation (figure 18a) As the free monomer

concentration is depleted to Cmono asymp SRF RF growth ceases and ThT fluorescence comes to a

plateau During the lag period detected by ThT no RFs or other pre-nucleation aggregates could

be detected with AFM (fig 18a(I) AFM panel aI) After the RF nucleation indicated by ThT

long straight fibrils can be readily observed with AFM (fig 18a(II) AFM panel aII)

Above the COC aggregation kinetics displays a different signature ThT fluorescence increases

immediately upon incubation and this corresponds with the formation of aO observed in AMF

images (fig 18b (I) AFM panel bI) With time the rate of ThT fluorescence increases slows

down which is consistent with a decreased driving force for aO formation Overall aOs tend to

polymerize into CFs as shown by AFM (fig 18b(II) AFM panel bII) For monomer

concentrations well above the COC aO concentrations reach the plateau as monomer

concentrations deplete to the COC and before RFs nucleate (fig 18b) The second increase in the

ThT fluorescence is associated with RF nucleation and growth (fig 18b(III) AFM panel b(III) -

b(V)) RFs grow until the monomeric substrate reaches the fibril solubility SRF It is important to

notice that AFM images of CFs in figure 18b AFM panels II and III show long curvilinear fibrils

and no or very few rigid fibrils while in panel IV and V the same sample shows considerable

58

shortening in curvilinear fibrils length and more rigid fibrils which is consistent with the

hypothesis that they undergo decay

(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)


Oligomer Concentration (a) Typical ThT kinetics for amyloid growth below the COC (here 20

Figure 18 Growth Kinetics and Aggregate Morphologies Below and Above Critical Oligomer

Concentration (Continued on Next Page)

59

uM HewL 500 mM NaCl pH 2 52 degC) During an initial lag period lasting from many hours to

days ThT emission remains flat The rapid upswing upon HewL RF nucleation eventually levels

off as RF growth depletes free monomer concentrations down to SRF AFM images obtained after

extended deposition periods yield only monomers during the lag period (aI) Following the

nucleation event RFs appear (aII) (b) ThT kinetics for amyloid growth above the COC (here

350 microM HewL 400 mM NaCl pH 2 52 degC) correlated to AFM images of aggregate

morphologies at the indicated time points (b I-V) An immediate increase in ThT kinetics

coincides with the formation of aOs and CFs (bI bII) As aOCF growth depletes monomer

concentrations back to COC a plateau phase is noticed followed by the nucleation and growth of

RFs The second slow down and plateau-like phase is due to slowing RFs growth rates as

monomers get depleted again

IV42 Amyloid Phase Diagram Permits Unbiased Test of On- Pathway Nucleated

Conformational Conversion vs Competitive Nucleated Polymerization

As it has been described in the introduction (see ID) there are several routes the RF

nucleus can form most notably via a random encounter of several monomers (NP) or through

the internal restructuring of early stage aggregates (NCC fig 19 right) The RF nucleation

mechanisms gain relevance in the light of the evidence that for numerous amyloidosis the early

stage oligomeric species are the main culprit while RF serve as a relatively harmless reservoir

for the denaturedunstable proteins Hence the RF nucleation and growth in the presence of

oligomeric species has a direct implication for the long term outcome of amyloid diseases As I

had shown in the earlier chapters our HewL system also transitions from early stage aOCF to

end stage RF for solution conditions above the COC This way besides providing a direct

rationale for the distinct kinetic signatures of amyloid self-assembly below and above the COC

the phase diagram represents a unique tool to inquire whether RF nucleation above the COC

proceeds via NP from monomer or involves nucleated conformation conversion (NCC) of

oligomers and curvilinear fibrils It is notable to point out that the growth kinetic curves alone

would not be able to discern between the two RF nucleation scenarios In both cases aOCF

form first as kinetically favored aggregated species which would correspond to the initial

60

increase in ThT Fluorescence (fig 19 left top and bottom) The nucleation and growth of RF

would be indicated by the second up rise in the ThT kinetics but there are no indications in the

fluorescence response on which route the RF nucleus formed Monitoring the aOCF after the RF

nucleation would not give a definite answer as well As we showed previously aOCF are the

metastable phase so if RF nucleation and subsequent growth proceed via monomers (NP)

aOCF are expected to decay once the monomeric substrate is depleted to concentrations below

the COC Hence for the NP mechanism aOCF would be off-pathway aggregates that would

slowly disassemble and serve as a steady source of monomers for RF growth until their complete

disappearance (buffering) If aOCF restructure into RF nuclei (NCC) then the RFs would

further grow from either monomers or aOCF so the early stage aggregates get further

restructured into nuclei get incorporated into the RF (on-pathway) andor decay (once the

monomeric substrate gets depleted below the COC)

An essential difference between NP and NCC scenarios for RF nucleation is the expected RFs

nucleation lag period behavior below and above the COC RF nucleation behavior has been

shown to carry information about the amyloid aggregation molecular mechanisms435574165166

At

fixed nucleation and elongation rates the lag period τlag follows a power law when is plotted

against total protein concentration with a fixed scaling exponent for NP or NP with secondary

nucleation (secondary nucleation existing nucleiRF can serve as catalytic surfaces for

nucleation by lowering the energy barrier for new nuclei formation)165166

Hence for a fixed salt

concentration below the COC the RF τlag should have a fixed scaling exponent but as the

HewL concentrations increase and aOCF start to form rapidly the τlag is expected to change

depending on the RF nucleation mechanism Distinguishing between the above RF nucleation

61

Figure 19 Schematic of Nucleated Conformational Conversion (NCC) vs Nucleated

Polymerization (NP) of Rigid Fibrils (RF) Aggregation kinetic curves and RF nucleation for a

sample above the COC (top) In nucleated conformational conversion (NCC) partially denatured

monomers first assemble into oligomers (aO) and curvilinear fibrils (CF) Either of these

aggregates subsequently spontaneously re-structures into a critical nucleus (NCC nucleus

formation marked by dotted line) that elongates into a rigid fibril (RF) Elongation might proceed

via monomer or oligomer addition The lag period for NCC arises from the low rates of aOCF

restructuring into an NCC nucleus (bottom) aOCF and RF form independentlyparallel to each

other RFs form via Nucleated Polymerization (NP) where partially denatured monomers (gray

curve) assemble by chance encounter into a critical nucleus (NP nucleus formation marked by

dotted vertical line) This nucleus subsequently grows via addition of partially unfolded

monomers into a rigid fibril (RF) The lag period in NP is associated with the low probability of

forming the initial nucleus Notice that kinetics alone would not be able to discern between these

two scenarios

lag periods behavior can be directly tested using the phase diagram at a fixed salt concentration

and increase in protein concentration leads to an increase in the amounts of aOsCF being formed

(fig 14 fig 17b)

62

Figure 20 Schematic of Anticipated Changes in Rigid Fibrils Lag Period Upon Crossing of

Critical Oligomer Concentration for Nucleated Polymerization (NP) Nucleated

Conformational Conversion (NCC) and Nucleated Polymerization (NP) with aO Buffering

Notice the same RF lag period scaling exponent for NP in absence of any aOCF (middle straight

line across the COC) RF lag period is expected to decrease as aOCF is increased RF lag period

in presence of off-pathway aOCF is expected to drop slightly in vicinity of COC following the

pure NP scaling behavior but eventually coming to the value for RF lag period at COC

In case RF nucleate via a random encounter of monomers (NP) in presence of aOCF (off-

pathway aggregates) the RF τlag is expected to follow the pure NP behavior in the vicinity of

COC since the driving force for aOCF is relatively modest and the effective total monomer

concentration is not changing during the RF τlag As the total protein concentration increases the

driving force for aOCF increases rapidly and the effective monomeric concentration decreases

rapidly during the lag period and eventually reaches the COC values Hence the RF τlag would

asymptotically approach the RF τlag at COC monomeric concentration as the total monomeric

concentration increases (fig 20)

63

If RF nucleate via internal restructuring of aOCF (NCC) then the probability of forming a

nucleus is strongly dependent on aOCF concentration As aOCF concentration increases one

would expect an increased probability of RF nucleation hence a shortening in the RFs lag

periods comparing to both pure NP and NP in presence of off-pathway aOCF (fig 20) Figure

20 is a schematic of the expected behavior of lag periods for RF nucleation for the case of NP

NP in presence of off-pathway aOCF and NCC across the COC

(a)

(b)

Figure 21 Lag Periods for Rigid Fibrils Nucleation below and above the Critical Oligomer

Concentration (a) Semi-log plot of HewL amyloid nucleation and growth kinetics just below

(orange) and above (purple) the COC for samples incubated at pH 2 T = 52 degC 450 mM NaCl

and at the indicated HewL concentrations The semi-logarithmic display highlights the aOCF

kinetics at the expense of the large ThT response following RF nucleation (b) Measured lag

period as a function of HewL monomer concentration extracted from a series of kinetics

measurements for the same set of solution and incubation conditions as shown in plot (a)

Since the COC delineates a sharp boundary one can measure the lag periods for RF nucleation

below the COC and compare it to those for RF nucleation in the presence of aOCF above the

COC This comparison allows one to establish if the presence of aOCFs shortens the lag period

64

for RFs strongly indicating that aOCFs are on-pathway or assist (aOCF surface serves as

preferred nucleation site) RF nucleation

In figure 21a the experimentally measured ThT kinetic curves for a series of HewL

concentrations are shown These concentrations span protein concentrations from just below to

well above the COC Each kinetic curve represent the average from at least three sample s

measured simultaneous in a plate reader and are consistent with earlier observations49138

The

semi-log display of the ThT kinetics is used to highlight the transition in growth kinetics Just

below and above the COC (50 microM for this specific salt concentration) the curves show similar

RFs lag period ThT fluorescence detects no aggregate formation below the COC at 35 microM

HewL and a small initial slope at 70 microM indicates the formation of aOs As the HewL

concentration is increased the initial slope rapidly increases and eventually at 350 microM HewL a

clear plateau indicating the reach of COC can be seen Surprisingly instead of decreasing or

reaching the value for HewL concentration at COC the lag periods for RFs nucleation slightly

increases Figure 21b summarizes the lag periods as a function of initial protein concentrations

for the same NaCl concentration as in figure 21b but for a wide protein concentration span It is

easy to notice that RFs lag periods in the vicinity of the COC are similar within experimental

error but increase as the protein concentration is increased The lack of shortening RF τlag in

presence of aOCF is good evidence that NP is the molecular mechanism of RFs nucleation on

both sides of the COC

We performed seeding experiments below the COC (fig 22) as additional confirmation that

HewL aOCF are incapable of conversion into RFs on the time scale of spontaneous RF

nucleation By adding preformed aOCF below the COC we also address the unlikely scenario

65

that we might have missed small populations of aOCF already present below the COC which in

turn might dominate nucleation rates below the COC

Figure 22 Lag Periods for Rigid

Fibrils Nucleation upon ldquoSeedingrdquo

with Oligomers and Curvilinear

Fibrils below the Critical Oligomer

Concentration

Semi-log plot of HewL RF nucleation

and growth kinetics below the COC

(38 microM HewL 400 mM NaCl)

without seeding (control) or after

seeding with isolated aOCF (purple)

or RF (orange)

As shown in figure 22 controls without addition of seeds undergo nucleated polymerization

Similarly addition of preformed and isolated RFs eliminates the lag period and induces

immediate fibril elongation In contrast addition of isolated aO CFs below the COC induces a

detectable increase in ThT response indicating that those concentrations are now well above any

potential level of ―contaminating oligomers we might not have picked up As expected below

the COC the aOCF undergo a slow decay Yet despite these highly elevated levels of aOCF

the lag period for RF nucleation yet again slightly increases just as observed in the presence of

aOCF above the COC This confirms that aOCF under all conditions do not undergo structural

conversion into RFs on a time scale competitive with NP of RF

66

V DISCUSSIONS AND CONCLUSIONS

Extracellular depositions of insoluble amyloid fibrils are the pathological hallmark of a

group of diseases called amyloidoses Alzheimer Parkinson and type II diabetes are just a few

examples of amyloidoses which are affecting millions of people worldwide Regardless of the

protein of origin amyloid fibrils have a set of structural characteristics that distinguish them as a

group Usually these are long straight unbranched proteinaceous fibrils that have a cross-β sheet

core easily recognizable by its X-ray diffraction pattern with a sharp reflection at 47 Å along the

fibril direction and a less sharp reflection at 10ndash11 Å perpendicular to the fibril direction This

cross-β sheet motif is commonly recognized by the benzothiazole dye Thioflavin T (ThT) and

when stained with Congo Red exhibits birefringenceunder cross polarized light Both in vitro

and in vivo experiments have shown that amyloid fibril formation can proceed via different

assembly pathways one of which involves early stage soluble intermediates called oligomers

These early stage oligomers are considered the main culprit of amyloidosis since they have been

shown to be the most toxic amyloid species Despite significant efforts the relationship between

the early stage oligomers and their late stage fibril has remained unclear There are currently two

dominant hypotheses of how early-stage oligomers are supplanted at late stage by the rigid

amyloid fibrils One model suggests that early stage aggregates are off-pathway but kinetically

favored with long rigid amyloid fibrils formed via nucleated polymerization (NP) from

67

monomers and independently from off-pathway oligomers The other model proposes that early

stage oligomers are on pathway kinetically favored pre-cursors of rigid fibrils that can undergo

internal restructuring to form the nuclei for rigid fibrils There have been reports supporting

either mechanism based on data from various amyloid and prion proteins Yet direct evidence for

one mechanism or the other is missing This is due in part to a lack of a coherent overview of

all the types of amyloid aggregates and their structure that are accessible to a system under well-

defined growth conditions In addition both the kinetics and morphological evolution of fibril

formation following either mechanism are qualitatively indistinguishable

In 2011 our lab identified and characterized the distinct aggregate populations formed by Hen

Egg White Lysozyme an amyloid forming protein under denaturing conditions (low pH high

temperature) Depending on the ionic strength HewL either formed long straight fibrils well-

defined globular oligomeric species that further polymerize into curvilinear fibrils or amorphous

aggregates52

Each self-assembly pathway had characteristic Static Light Scattering (SLS)

kinetics signatures and distinct aggregate populations observed with Dynamic Light Scattering

(DLS) and Atomic Forces Microscopy (AFM) In 2013 using ThT and Fourier Transform

Infrared Spectroscopy (FTIR) as structural probes we reported that both long rigid fibrils and

oligomeric species display features indicative of their underlying amyloid structures ThT also

indicated that polymerization of oligomers into curvilinear fibrils proceeds without significant

restructuring of oligomers Hence oligomers and curvilinear fibrils represent amyloid species

distinct from rigid fibrils We also showed that ThT has orders of magnitude higher affinity for

rigid fibrils than for oligomeric species and that ThT responses closely matched the SLS kinetics

signature

68

One of my research objectives was to map out the protein- and salt-concentration phase space for

amyloid fibril formation under conditions of fixed pH and incubation temperature Specifically I

set out to determine the combinations of protein and salt concentrations that resulted in the

growth of either rigid fibrils or the formation of oligomers and curvilinear fibrils Towards that

end I used aggregation kinetics spectroscopy and high-resolution imaging to ascertain which

type of amyloid aggregates had emerged This kinetic phase diagram of amyloid assembly

spanned HewL concentrations from 6 μM to 14 mM and NaCl concentrations from 50minus800 mM

As outlined above in section III33 my data indicate that long rigid fibrils (RF) are the

thermodynamically stable state and they are the only aggregate state that can form in the low

monomersalt region of the phase diagram Oligomers and their curvilinear fibrils (aOCF) in

turn are the kinetically favored amyloid state at high monomersalt concentrations of the phase

space The onset of oligomer formation is delineated by a sharp solubility boundary with strong

similarities to the critical micelle concentration in surfactant solutions hence we called this

boundary the Critical Oligomer Concentration (COC) Equally important I have shown that the

oligomerscurvilinear fibrils represent a metastable phase ie rigid fibrils seeded above the

COC readily grew and eventually dissolved existing oligomers and curvilinear fibrils I also

mapped out the kinetic transition for the onset of precipitation at very high protein andor salt

concentrations Our lab had previously considered these precipitates to be structureless

amorphous aggregates Using FTIR I showed instead that these kinetically trapped aggregates

already have the spectroscopic signatures characteristic of the amyloid oligomers formed at

lower proteinsalt concentrations In collaboration with Dr Jeremy Schmit from Kansas State

we established that the salt dependence of the COC was reproduced with surprising accuracy

using a simplified colloidal model of charge effects on amyloid oligomer stability With

69

additional modifications Dr Schmit could extend this model further to reproduce the transition

to the kinetically trapped precipitates

As noted in the introduction one of the persistent questions in the field concerns the

relation between early-stage amyloid oligomers and late-stage rigid fibrils Are oligomers on-

pathway to rigid fibril formation (nucleated conformational conversion NCC) or are they off-

pathway competitors to rigid fibril growth with the latter emerging via nucleated

polymerization The above phase diagram gave us a unique tool to address this conundrum

Below the COC ie in the region of the phase diagram where only RF can exist RF form via

nucleated polymerization Above the COC however their presence in the late stages of

aggregation could come about by either on-pathway NCC or independent NP According to our

kinetic measurement above the COC aOCF form immediately upon incubation thereby

depleting the monomer substrate for RF nucleation via NP Once the monomers are depleted

down to the COC the aOCF driving force is zero and they stop growing The RFs solubility in

contrast is much lower As a result they can nucleate and grow above and below the COC

however the presence of different concentrations of aOCF above the COC provides a unique

way to distinguish between off-pathway nucleation of RFs from monomers vs on-pathway

nucleation from oligomers If RF nucleation requires on-pathway oligomers increasing

concentrations of aOCF should decrease the lag period especially at high concentrations of

oligomers In contrast the depletion of monomers by aOCF down to the COC would buffer

monomer concentrations and therefore clamp monomer concentrations available for RF

nucleation to their value near the COC As outlined above I tested these predictions by

measuring the lag periods for RF nucleation below to well above the COC These measurements

provide good evidence that RF form via nucleated polymerization regardless of the presence of

70

aOCF (see Section III42) Moreover ―seeding with the metastable aOCF below the COC did

not decrease RFs lag period which provided yet another strong hint that aOCF are incapable of

restructuring into RF nuclei (at least not in the time span of our kinetic measurements)

71

REFERENCES

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Med 337 898ndash909 (1997)

2 Rambaran R N amp Serpell L C Amyloid fibrils Prion (2008)

3 Buxbaum J N amp Linke R P A Molecular History of the Amyloidoses J Mol Biol 421

142ndash159 (2012)

4 Alzheimerlsquos Association 2016 Alzheimerlsquos Disease Facts and Figures Alzheimerrsquos Dement

12 (2016)


11 (2015)

6 Ruberg F L amp Berk J L Transthyretin (TTR) cardiac amyloidosis Circulation 126 1286ndash

1300 (2012)

7 Virchow R L C Nouvelles Observations sur la substance animale analogue a la cellulose

vegetale Monit Hopiteaux 1199 (1853)

8 Friedreich N amp Kekuleacute A Zur amyloidfrage Virchows Arch 16 50ndash65 (1859)

9 Divry P amp Florkin M Sur les proprietes optiques de llsquoamyloide CR Soc Biol 97 1808ndash

1810 (1927)

10 Wolman M amp Bubis J J The cause of the green polarization color of amyloid stained with

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72

11 Sipe J D amp Cohen A S Review History of the Amyloid Fibril J Struct Biol 130 88ndash98

(2000)

12 Biancalana M Makabe K Koide A amp Koide S Molecular Mechanism of Thioflavin-T

Binding to the Surface of β-Rich Peptide Self-Assemblies J Mol Biol 385 1052ndash1063

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13 Biancalana M amp Koide S Molecular mechanism of Thioflavin-T binding to amyloid

fibrils Biochim Biophys Acta BBA - Proteins Proteomics 1804 1405ndash1412 (2010)

14 Khurana R et al Mechanism of thioflavin T binding to amyloid fibrils J Struct Biol 151

229ndash238 (2005)

15 Sabateacute R Lascu I amp Saupe S J On the binding of Thioflavin-T to HET-s amyloid fibrils

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16 Cohen A S amp Calkins E Electron microscopic observations on a fibrous component in

amyloid of diverse origins Nature 183 1202ndash1203 (1959)

17 Eans E D amp Glenner G G X-RAY DIFFRACTION STUDIES ON AMYLOID

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18 Glenner G G Terry W Harada M Isersky C amp Page D Amyloid Fibril Proteins

Proof of Homology with Immunoglobulin Light Chains by Sequence Analyses Science 172

1150ndash1151 (1971)

19 Glenner G G et al Creation of Amyloidlsquo Fibrils from Bence Jones Proteins in vitro

Science 174 712ndash714 (1971)

20 Benditt E P amp Eriksen N Chemical Classes of Amyloid Substance Am J Pathol 65

231ndash252 (1971)

73

21 I K M R amp E S In vitro synthesis of amyloidlsquofibrils from insulin calcitonin and

parathormone Isr J Med Sci 12 1137ndash1140 (1976)

22 Connors L H Shirahama T Skinner M Fenves A amp Cohen A S In vitro formation of

amyloid fibrils from intact β2-microglobulin Biochem Biophys Res Commun 131 1063ndash

1068 (1985)

23 Castantildeo E M et al In vitro formation of amyloid fibrils from two synthetic peptides of

different lengths homologous to alzheimerlsquos disease β-protein Biochem Biophys Res

Commun 141 782ndash789 (1986)

24 Kirschner D A et al Synthetic peptide homologous to beta protein from Alzheimer disease

forms amyloid-like fibrils in vitro Proc Natl Acad Sci 84 6953ndash6957 (1987)

25 Gustavsson Aring Engstroumlm U amp Westermark P Normal transthyretin and synthetic

transthyretin fragments from amyloid-like fibrils in vitro Biochem Biophys Res Commun

175 1159ndash1164 (1991)

26 Glenner G G Reprint of Alzheimerlsquos disease Initial report of the purification and

characterization of a novel cerebrovascular amyloid proteinlsquo Biochem Biophys Res

Commun 425 534ndash539 (2012)

27 Prusiner S B Novel proteinaceous infectious particles cause scrapie Science 216 136ndash144

(1982)

28 Chiti F et al Designing conditions for in vitro formation of amyloid protofilaments and

fibrils Proc Natl Acad Sci 96 3590ndash3594 (1999)

29 Kelly J W The alternative conformations of amyloidogenic proteins and their multi-step

assembly pathways Curr Opin Struct Biol 8 101ndash106 (1998)

74

30 Dobson C M Protein misfolding evolution and disease Trends Biochem Sci 24 329ndash332

(1999)

31 Dobson C M Protein folding and misfolding Nature 426 884ndash890 (2003)

32 Aso Y Shiraki K amp Takagi M Systematic Analysis of Aggregates from 38 Kinds of Non

Disease-Related Proteins Identifying the Intrinsic Propensity of Polypeptides to Form

Amyloid Fibrils Biosci Biotechnol Biochem 71 1313ndash1321 (2007)

33 Sunde M et al Common core structure of amyloid fibrils by synchrotron X-ray diffraction

J Mol Biol 273 729ndash739 (1997)

34 Fraser P E Nguyen J T Surewicz W K amp Kirschner D A pH-dependent structural

transitions of Alzheimer amyloid peptides Biophys J 60 1190ndash1201 (1991)

35 Benzinger T L et al Propagating structure of Alzheimerlsquos β-amyloid (10ndash35) is parallel β-

sheet with residues in exact register Proc Natl Acad Sci 95 13407ndash13412 (1998)

36 Balbirnie M Grothe R amp Eisenberg D S An amyloid-forming peptide from the yeast

prion Sup35 reveals a dehydrated β-sheet structure for amyloid Proc Natl Acad Sci 98

2375ndash2380 (2001)

37 Nelson R et al Structure of the cross-β spine of amyloid-like fibrils Nature 435 773ndash778

(2005)

38 Nelson R amp Eisenberg D Structural Models of Amyloid‐Like Fibrils in Advances in

Protein Chemistry 73 235ndash282 (Elsevier 2006)

39 Sawaya M R et al Atomic structures of amyloid cross-β spines reveal varied steric zippers

Nature 447 453ndash457 (2007)

40 Fitzpatrick A W P et al Atomic structure and hierarchical assembly of a cross- amyloid

fibril Proc Natl Acad Sci 110 5468ndash5473 (2013)

75

41 Petkova A T et al A structural model for Alzheimerlsquos beta-amyloid fibrils based on

experimental constraints from solid state NMR Proc Natl Acad Sci 99 16742ndash16747

(2002)

42 Tycko R Molecular structure of amyloid fibrils insights from solid-state NMR Q Rev

Biophys 39 1 (2006)

43 Arosio P Knowles T P J amp Linse S On the lag phase in amyloid fibril formation Phys

Chem Chem Phys 17 7606ndash7618 (2015)

44 Sawyer E B Claessen D Gras S L amp Perrett S Exploiting amyloid how and why

bacteria use cross-β fibrils Biochem Soc Trans 40 728ndash734 (2012)

45 Smith J Mowles A Mehta A amp Lynn D Looked at Life from Both Sides Now Life 4

887ndash902 (2014)

46 Lashuel H A Lai Z amp Kelly J W Characterization of the transthyretin acid denaturation

pathways by analytical ultracentrifugation implications for wild-type V30M and L55P

amyloid fibril formation Biochemistry (Mosc) 37 17851ndash17864 (1998)

47 Stine Jr W B et al The nanometer-scale structure of amyloid-β visualized by atomic force

microscopy J Protein Chem 15 193ndash203 (1996)

48 Harper J D Wong S S Lieber C M amp Lansbury P T J Observation of metastable Aβ

amyloid protofibrils by atomic force microscopy Chem Biol 4 119ndash125 (1997)

49 Walsh D M et al Naturally secreted oligomers of amyloid β protein potently inhibit

hippocampal long-term potentiation in vivo Nature 416 535ndash539 (2002)

50 Lambert M P et al Diffusible nonfibrillar ligands derived from Aβ1ndash42 are potent central

nervous system neurotoxins Proc Natl Acad Sci 95 6448ndash6453 (1998)

76

51 Ross C A amp Poirier M A Protein aggregation and neurodegenerative disease Nat Med

10 S10ndashS17 (2004)

52 Hill S E Miti T Richmond T amp Muschol M Spatial Extent of Charge Repulsion

Regulates Assembly Pathways for Lysozyme Amyloid Fibrils PLoS ONE 6 e18171 (2011)

53 Modler A Gast K Lutsch G amp Damaschun G Assembly of Amyloid Protofibrils via

Critical OligomersmdashA Novel Pathway of Amyloid Formation J Mol Biol 325 135ndash148

(2003)

54 Gosal W S et al Competing Pathways Determine Fibril Morphology in the Self-assembly

of β2-Microglobulin into Amyloid J Mol Biol 351 850ndash864 (2005)

55 Lorenzen N et al The Role of Stable α-Synuclein Oligomers in the Molecular Events

Underlying Amyloid Formation J Am Chem Soc 136 3859ndash3868 (2014)

56 Eichner T amp Radford S E Understanding the complex mechanisms of β2-microglobulin

amyloid assembly β2-microglobulin fibrillogenesis at physiological pH FEBS J 278 3868ndash

3883 (2011)

57 Malisauskas M et al Intermediate amyloid oligomers of lysozyme Is their cytotoxicity a

particular case or general rule for amyloid Biochem Mosc 71 505ndash512 (2006)

58 Serio T R et al Nucleated conformational conversion and the replication of conformational

information by a prion determinant Science 289 1317ndash1321 (2000)

59 Lee J Culyba E K Powers E T amp Kelly J W Amyloid-β forms fibrils by nucleated

conformational conversion of oligomers Nat Chem Biol 7 602ndash609 (2011)

60 Shammas S L et al A mechanistic model of tau amyloid aggregation based on direct

observation of oligomers Nat Commun 6 7025 (2015)

77

61 Cremades N et al Direct Observation of the Interconversion of Normal and Toxic Forms of

α-Synuclein Cell 149 1048ndash1059 (2012)

62 Scheibel T Bloom J amp Lindquist S L The elongation of yeast prion fibers involves

separable steps of association and conversion Proc Natl Acad Sci U S A 101 2287ndash

2292 (2004)

63 Reixach N Deechongkit S Jiang X Kelly J W amp Buxbaum J N Tissue damage in the

amyloidoses Transthyretin monomers and nonnative oligomers are the major cytotoxic

species in tissue culture Proc Natl Acad Sci U S A 101 2817ndash2822 (2004)

64 Glabe C G amp Kayed R Common structure and toxic function of amyloid oligomers

implies a common mechanism of pathogenesis Neurology 66 S74ndashS78 (2006)

65 Lesneacute S et al A specific amyloid-β protein assembly in the brain impairs memory Nature

440 352ndash357 (2006)

66 Gharibyan A L et al Lysozyme Amyloid Oligomers and Fibrils Induce Cellular Death via

Different ApoptoticNecrotic Pathways J Mol Biol 365 1337ndash1349 (2007)

67 Jakhria T et al 2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt

Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes J

Biol Chem 289 35781ndash35794 (2014)

68 Tipping K W van Oosten-Hawle P Hewitt E W amp Radford S E Amyloid Fibres Inert

End-Stage Aggregates or Key Players in Disease Trends Biochem Sci 40 719ndash727 (2015)

69 Gillam J E amp MacPhee C E Modelling amyloid fibril formation kinetics mechanisms of

nucleation and growth J Phys Condens Matter 25 373101 (2013)

70 Chiti F Stefani M Taddei N Ramponi G amp Dobson C M Rationalization of the

effects of mutations on peptide and protein aggregation rates Nature 424 805ndash808 (2003)

78

71 Zhao D amp Moore J S Nucleationndashelongation a mechanism for cooperative

supramolecular polymerization Org Biomol Chem 1 3471ndash3491 (2003)

72 Knowles T P J et al An Analytical Solution to the Kinetics of Breakable Filament

Assembly Science 326 1533ndash1537 (2009)

73 Cohen S I A et al Nucleated polymerization with secondary pathways I Time evolution

of the principal moments J Chem Phys 135 065105 (2011)

74 Cohen S I Vendruscolo M Dobson C M amp Knowles T P Nucleated polymerization

with secondary pathways II Determination of self-consistent solutions to growth processes

described by non-linear master equations J Chem Phys 135 065106 (2011)


with secondary pathways III Equilibrium behavior and oligomer populations J Chem

Phys 135 065107 (2011)

76 Cohen S I A Vendruscolo M Dobson C M amp Knowles T P J From Macroscopic

Measurements to Microscopic Mechanisms of Protein Aggregation J Mol Biol 421 160ndash

171 (2012)

77 Garcia G A Cohen S I A Dobson C M amp Knowles T P J Nucleation-conversion-

polymerization reactions of biological macromolecules with prenucleation clusters Phys

Rev E 89 (2014)

78 Morris A M Watzky M A amp Finke R G Protein aggregation kinetics mechanism and

curve-fitting A review of the literature Biochim Biophys Acta BBA - Proteins Proteomics

1794 375ndash397 (2009)

79 Oosawa F Asakura S Hotta K Imai N amp Ooi T G-F Transformation of Actin as a

Fibrous Condensation J Polym Sci 37 232ndash336 (1959)

79

80 Oosawa F amp Kasai M A theory of linear and helical aggregations of macromolecules J

Mol Biol 4 10ndash21 (1962)

81 Kasai M Asakura S amp Oosawa F The cooperative nature of G-F transformation of actin

Biochim Biophys Acta 57 22ndash31 (1962)

82 Oosawa F Size distribution of protein polymers J Theor Biol 27 69ndash86 (1970)

83 La Mer V K Nucleation in Phase Transitions Ind Engeneering Chem 44 1270ndash1277

(1952)

84 Kaschiev D Nucleation (Butterworth-Heinemann 2000)

85 Hofrichter J Ross P D amp Eaton W A Kinetics and mechanism of deoxyhemoglobin S

gelation a new approach to understanding sickle cell disease Proc Natl Acad Sci 71

4864ndash4868 (1974)

86 Ferrone F A Hofrichter J Sunshine H R amp Eaton W A Kinetic studies on photolysis-

induced gelation of sickle cell hemoglobin suggest a new mechanism Biophys J 2 361ndash380

(1980)

87 Wegner A amp Engel J Kinetics of the Cooperative Association of Aactin to Actin

Filaments Biophys Chem 3 215ndash225 (1975)

88 Tobacman L S amp Korn E D The kinetics of actin nucleation and polymerization J Biol

Chem 258 3207ndash14 (1983)

89 Frieden C amp Goddette D W Polymerization of Actin and Actin-like Systems Evaluation

of the Time Course of Polymerization in Relation to the Mechanismt Biochemistry (Mosc)

22 5836ndash5843 (1983)

80

90 Firestone M P De Levie R amp Rangarajan S K On One-dimensional Nucleation and

Growth of Livinglsquo Polymers I Homogeneous Nucleation J Theor Biol 104 535ndash552

(1983)

91 Goldstein R F amp Stryer L Cooperative Polymerization Reactions Analytical

Approximations Numerical Examples and Experimental Strategy Biophys J 50 583ndash599

(1986)

92 Watzky M A amp Finke R G Transition Metal Nanocluster Formation Kinetic and

Mechanistic Studies A New Mechanism When Hydrogen Is the Reductant Slow

Continuous Nucleation and Fast Autocatalytic Surface Growth J Am Chem Soc 119

10382ndash10400 (1997)

93 Thusius D Dessen P amp Jallon J-M Mechanism of Bovine Liver Glutamate

Dehydrogenase Self-association I Kinetic Evidence for a Random Association of Polymer

Chains J Mol Biol 92 413ndash432 (1975)

94 Thusius D Mechanism of bovine liver glutamate dehydrogenase self-assembly II

Simulation of relaxation spectra for an open linear polymerization proceeding via a

sequential addition of monomer units J Mol Biol 94 367ndash383 (1975)

95 Wegner A amp Savko P Fragmentation of actin filaments Biochemistry (Mosc) 21 1909ndash

1913 (1982)

96 Prusiner S B Molecular Biology of Prion Diseases Science 252 1515ndash1522 (1991)

97 Griffith J S Nature of the Scrapie Agent Self-replication and Scrapie Nature 215 1043ndash

1044 (1967)

98 Jarrett J T amp Lansbury P T Seeding one-dimensional crystallizationlsquo of amyloid a

pathogenic mechanism in Alzheimerlsquos disease and scrapie Cell 73 1055ndash1058 (1993)

81

99 Come J H Fraser P E amp Lansbury P T A kinetic model for amyloid formation in the

prion diseases importance of seeding Proc Natl Acad Sci 90 5959ndash5963 (1993)

100 Lansbury P T J amp Cauchey B The chemistry of scrapie infection implicationsof the

ice 9lsquo metaphor Chem Biol 2 1ndash5 (1995)

101 Lansbury P T amp Lashuel H A A century-old debate on protein aggregation and

neurodegeneration enters the clinic Nature 443 774ndash779 (2006)

102 Harper J D amp Lansbury P T Models of Amyloid Seeding in Alzheimerlsquos Disease and

Scrapie Mechanistic Truths and Physiological Consequences of the Time-Dependent

Solubility of Amyloid Proteins Annu Rev Biochem 66 385ndash407 (1997)

103 Kundu B et al Nucleation-dependent conformational conversion of the Y145Stop

variant of human prion protein Structural clues for prion propagation Proc Natl Acad Sci

100 12069ndash12074 (2003)

104 Dumetz A C Chockla A M Kaler E W amp Lenhoff A M Effects of pH on proteinndash

protein interactions and implications for protein phase behavior Biochim Biophys Acta BBA

- Proteins Proteomics 1784 600ndash610 (2008)

105 Dumetz A C Chockla A M Kaler E W amp Lenhoff A M Protein Phase Behavior

in Aqueous Solutions Crystallization Liquid-Liquid Phase Separation Gels and

Aggregates Biophys J 94 570ndash583 (2008)

106 Bolisetty S Harnau L Jung J amp Mezzenga R Gelation Phase Behavior and

Dynamics of β-Lactoglobulin Amyloid Fibrils at Varying Concentrations and Ionic

Strengths Biomacromolecules 13 3241ndash3252 (2012)

107 Yoshimura Y et al Distinguishing crystal-like amyloid fibrils and glass-like amorphous

aggregates from their kinetics of formation Proc Natl Acad Sci 109 14446ndash14451 (2012)

82

108 So M et al Supersaturation-Limited and Unlimited Phase Spaces Compete to Produce

Maximal Amyloid Fibrillation near the Critical Micelle Concentration of Sodium Dodecyl

Sulfate Langmuir 31 9973ndash9982 (2015)

109 Whitelam S amp Jack R L The Statistical Mechanics of Dynamic Pathways to Self-

Assembly Annu Rev Phys Chem 66 143ndash163 (2015)

110 Gazit E Mechanisms of amyloid fibril self-assembly and inhibition Model short

peptides as a key research tool FEBS J 272 5971ndash5978 (2005)



112 Whitelam S Hedges L O amp Schmit J D Self-Assembly at a Nonequilibrium Critical

Point Phys Rev Lett 112 (2014)

113 Zhang J amp Liu X Y Effect of proteinndashprotein interactions on protein aggregation

kinetics J Chem Phys 119 10972 (2003)

114 Jacobs W M Oxtoby D W amp Frenkel D Phase separation in solutions with specific

and nonspecific interactions J Chem Phys 140 204109 (2014)

115 Schmit J D Ghosh K amp Dill K What Drives Amyloid Molecules To Assemble into

Oligomers and Fibrils Biophys J 100 450ndash458 (2011)

116 Foderagrave V Zaccone A Lattuada M amp Donald A M Electrostatics Controls the

Formation of Amyloid Superstructures in Protein Aggregation Phys Rev Lett 111 (2013)

117 Foley J et al Structural fingerprints and their evolution during oligomeric vs oligomer-

free amyloid fibril growth J Chem Phys 139 121901 (2013)

83

118 Marshall K E et al Hydrophobic Aromatic and Electrostatic Interactions Play a

Central Role in Amyloid Fibril Formation and Stability Biochemistry (Mosc) 50 2061ndash

2071 (2011)

119 Hou L et al Solution NMR studies of the Aβ (1-40) and Aβ (1-42) peptides establish

that the Met35 oxidation state affects the mechanism of amyloid formation J Am Chem

Soc 126 1992ndash2005 (2004)

120 Sulatskaya A I Kuznetsova I M amp Turoverov K K Interaction of Thioflavin T with

Amyloid Fibrils Stoichiometry and Affinity of Dye Binding Absorption Spectra of Bound

Dye J Phys Chem B 115 11519ndash11524 (2011)



122 Kong J amp Yu S Fourier Transform Infrared Spectroscopic Analysis of Protein

Secondary Structures Acta Biochim Biophys Sin 39 549ndash559 (2007)

123 Barth A Infrared spectroscopy of proteins Biochim Biophys Acta BBA - Bioenerg

1767 1073ndash1101 (2007)

124 Chirgadze Y N amp Nevskaya N A Infrared spectra and resonance interaction of amide-

I vibration of the antiparallel-chain pleated sheet Biopolymers 15 607ndash625 (1976)


I vibration of the parallel-chain pleated sheet Biopolymers 15 627ndash636 (1976)

126 Nevskaya N A amp Chirgadze Y N Infrared spectra and resonance interactions of

amide-I and II vibrations of α-helix Biopolymers 15 637ndash648 (1976)

127 Bandekar J amp Krimm S Vibrational analysis of peptides polypeptides and proteins

Characteristic amide bands of beta-turns Proc Natl Acad Sci 72 774ndash777 (1979)

84

128 Krimm S Vibrational analysis of conformation in peptides polypeptides and proteins

Biopolymers 22 217ndash225 (1983)

129 Bandekar J amp Krimm S Normal mode spectrum of the parallel-chain β-sheet


130 Cerf E et al Antiparallel β-sheet a signature structure of the oligomeric amyloid β-

peptide Biochem J 421 415ndash423 (2009)

131 Celej M S et al Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive

antiparallel β-sheet structure Biochem J 443 719ndash7126 (2012)

132 Chen S W et al Structural characterization of toxic oligomers that are kinetically

trapped during α-synuclein fibril formation Proc Natl Acad Sci 112 E1994ndashE2003

(2015)

133 Fabian H et al Early Stages of Misfolding and Association of β2-Microglobulin

Insights from Infrared Spectroscopy and Dynamic Light Scattering Biochemistry (Mosc)

47 6895ndash6906 (2008)

134 Frare E et al Characterization of Oligomeric Species on the Aggregation Pathway of

Human Lysozyme J Mol Biol 387 17ndash27 (2009)

135 Mossuto M F et al The Non-Core Regions of Human Lysozyme Amyloid Fibrils

Influence Cytotoxicity J Mol Biol 402 783ndash796 (2010)

136 Frare E et al Identification of the Core Structure of Lysozyme Amyloid Fibrils by

Proteolysis J Mol Biol 361 551ndash561 (2006)

137 Fleming A On a Remarkable Bacteriolytic Element Found in Tissues and Secretions

Proc R Soc 93 (1922)

85

138 Jolles P Lysozymes in Proceedings of the Biochemical Society (Biochemical Journal

1986)

139 Jolles J Jauregui-Adell J BEernier I amp Jolles P La Structure Chmique du Lysozyme

de Blanc dlsquoOeuf de Poule Etude Detailllee Biochim Biophys ACTA 78 668ndash689 (1963)

140 Blake C C F et al Structure of Hen Egg-White Lysozyme A Three-dimensional

Fourier Synthesis at 2 Aring Resolution Nature 206 757ndash761 (1965)

141 Blake C C F Mair G A North A C T Phillips D C amp Sarma V R On the

conformation of hen egg-white lysozyme molecule Proc R Soc B 167 365ndash377

142 Blake C C F et al Crystallographic Studies of the Activity of Hen Egg-White

Lysozyme Proc R Soc 167 378ndash388 (1967)

143 Chipman D M amp Sharon N Mechanism of lysozyme action Science 165 454ndash465

(1969)

144 Rees A R amp Offord R E X-ray crystallography and the amino acid sequence of

lysozyme Biochem J 130 965ndash968 (1972)

145 Castellvi A amp Juanhuix J Room temperature X-ray diffraction of tetragonal HEWL

First data set (031 MGy) Be Publ doi102210pdb5l9jpdb

146 Ponikovaacute S et al Lysozyme stability and amyloid fibrillization dependence on

Hofmeister anions in acidic pH JBIC J Biol Inorg Chem 20 921ndash933 (2015)

147 Arnaudov L N amp de Vries R Thermally Induced Fibrillar Aggregation of Hen Egg

White Lysozyme Biophys J 88 515ndash526 (2005)

148 Kelly S M amp Price N C The use of circular dichroism in the investigation of protein

structure and function Curr Protein Pept Sci 1 349ndash384 (2000)

86

149 Pepys M B et al Human lysozyme gene mutations cause hereditary systemic

amyloidosis Nature 362 553ndash557 (1993)

150 Gillmore J D Booth D R Madhoo S Pepys M B amp Hawkins P N Hereditary

renal amyloidosis associated with variant lysozyme in a large English family Nephrol Dial

Transplant 14 2639ndash2644 (1999)

151 Jean E et al A new family with hereditary lysozyme amyloidosis with gastritis and

inflammatory bowel disease as prevailing symptoms Gastroenterology 14 PMC4171570

(2014)

152 Kumita J R et al Impact of the native-state stability of human lysozyme variants on

protein secretion by Pichia pastoris FEBS J 273 711ndash720 (2006)

153 Kumita J R et al Disease-related amyloidogenic variants of human lysozyme trigger

the unfolded protein response and disturb eye development in Drosophila melanogaster

FASEB J 26 192ndash202 (2012)

154 Frare E Polverino de Laureto P Zurdo J Dobson C M amp Fontana A A Highly

Amyloidogenic Region of Hen Lysozyme J Mol Biol 340 1153ndash1165 (2004)

155 Krebs M R et al Formation and seeding of amyloid fibrils from wild-type hen

lysozyme and a peptide fragment from the β-domain J Mol Biol 300 541ndash549 (2000)

156 Booth D R et al Instability Unfolding and Aggregation of Human Lysozyme Variants

Underlying Amyloid Fibrillogenesis NATURE 385 787ndash793 (1997)

157 Sasahara K Yagi H Naiki H amp Goto Y Heat-induced Conversion of β2-

Microglobulin and Hen Egg-white Lysozyme into Amyloid Fibrils J Mol Biol 372 981ndash

991 (2007)

87

158 Zandomeneghi G Krebs M R H McCammon M G amp Faumlndrich M FTIR reveals

structural differences between native β-sheet proteins and amyloid fibrils Protein Sci 13

3314ndash3321 (2009)

159 Rosenberger F Vekilov P G Muschol M amp Thomas R B Nucleation and

crystallization of globular proteins - what we know and what is missing J Cryst Growth

1668 1ndash27 (1996)

160 Narayanan J amp Liu X Y Protein Interactions in Undersaturated and Supersaturated

Solutions A Study Using Light and X-Ray Scattering Biophys J 84 523ndash532 (2003)

161 Bouchard M Zurdo J Nettleton E J Dobson C M amp Robinson C V Formation of

insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

Protein Sci 9 1960ndash1967 (2000)

162 Kroes-Nijboer A Venema P Bouman J amp van der Linden E Influence of Protein

Hydrolysis on the Growth Kinetics of β-lg Fibrils Langmuir 27 5753ndash5761 (2011)

163 Mulaj M Foley J amp Muschol M Amyloid Oligomers and Protofibrils but Not

Filaments Self-Replicate from Native Lysozyme J Am Chem Soc 136 8947ndash8956

(2014)

164 Miti T Mulaj M Schmit J D amp Muschol M Stable Metastable and Kinetically

Trapped Amyloid Aggregate Phases Biomacromolecules 16 326ndash335 (2015)

165 Meisl G et al Molecular mechanisms of protein aggregation from global fitting of

kinetic models Nat Protoc 11 252ndash272 (2016)

166 Eden K Morris R Gillam J MacPhee C E amp Allen R J Competition between

Primary Nucleation and Autocatalysis in Amyloid Fibril Self-Assembly Biophys J 108

632ndash643 (2015)

88

167 Miller L M Bourassa M W amp Smith R J FTIR spectroscopic imaging of protein

aggregation in living cells Biochim Biophys Acta BBA - Biomembr 1828 2339ndash2346

(2013)

168 Baker M J et al Using Fourier transform IR spectroscopy to analyze biological

materials Nat Protoc 9 1771ndash1791 (2014)

89

APPENDICES

Appendix A Critical Oligomer Concentration and Ppt Solubility Boundary

Theoretical Model

In the part a of the Appendix I am giving the detailed description of the theoretical

model of the cooperative COC transition presented earlier and being a part of the 2015

Biomacromolecules article


Concentration Boundary

In order to replicate the ionic strength dependence of the COC we developed a

quantitative model of the electrostatic energy cost of confining charged monomers within the

volume of an amyloid oligomer Assuming rapid monomer-oligomer interconversion the

concentration of oligomers is related to the monomer concentration by an equation taking the

form an nth

order reaction equilibrium ie

(1)

Here c1 and cn are the monomer and oligomer concentrations in molL respectively and Fn is the

coalescence free energy of the oligomer discussed below We chose n=8 for the number of

monomers in an oligomer This number is based on our previous experimental measurements of

the narrow distribution of lysozyme oligomer volumes under these condition This equation

predicts a very sharp transition with the oligomer concentration rising as the 8th

power of the

90

monomer concentration (Fig A11a) To define a critical oligomer concentration we use the

criterion that equal amounts of protein are in the monomer and oligomer states which

yields a critical concentration

(2)

Given the steep dependence of cn on c1 the precise choice of definition for the phase boundary is

not critical We are interested in how the COC depends on salt concentration We split the

oligomerization free energy into two parts where Fnon-ES is a non-

electrostatic contribution that is independent of salt concentration The electrostatic term

is the difference in the electrostatic free energy between n like-charged

monomers condensed into the volume of one oligomer vs n widely separated monomers (see

schematic in Fig 6b) The critical concentration then takes the form

(3)

where A is a parameter obtained from fitting the experimentally measured COC

Fn and F1 are computed by approximating the protein and oligomer as charged spheres as

described in the methods section We do not expect that the simplifying assumption of a

uniformly distributed surface charge significantly affects our results First the strong positive

charge of lysozyme at pH 2 limits contributions from spatial variations in charge distribution

Secondly more than half of the electrostatic contribution to the self-assembly energy comes

from the distortion of their Debye screening layers 39

This latter contribution is not sensitive to

D Finally the

product of actual oligomer assembly will have a high (8-fold) symmetry The calculated COClsquos

were fit to the experimental data using

as the only adjustable

91

parameter The resulting model predictions of the COC are shown in Fig 4 as a solid purple line

through the experimental transition values The model replicates the experimental values

remarkably well despite the significant number of simplifying assumptions Some minor

systematic deviations are noticeable in the semi-log plot of the phase diagram for high salt and

low protein concentrations

(a)

(b)

(c)

Figure A1 Colloidal Model for Critical Oligomer Concentration and Onset of Oligomer

Precipitation (a) Plot of monomer and oligomer concentration vs total protein concentration

Ctot for a fixed salt concentration based on Eqn (1) Notice the highly cooperative transition to

oligomer formation with increasing Ctot which replicates the sharp COC boundary in our

experimental data The COC defined via c1 = n cn is close to the sharp upturn in cn (b) The

colloidal model for amyloid oligomer formation (COC) considers a quasi-equilibrium between

charged HEWL monomers and their oligomers (the color change from monomers to

oligomers in this schematic does imply an underlying structural change) The model accounts

for the salt-dependent cost in free energy of confining n monomers into an n-meric oligomer

Monomers and oligomers are surrounded by an orange cloud indicating the diffusive

screening layer of salt ions (c) Schematic of the colloidal model used to calculate the

contributions of electrostatic repulsion of oligomers condensing into precipitates The short

range of the Debye layer compared to the size of the precipitates implies that the electrostatic

contributions to the free energy are essentially the same for CFs and O-Ppts This

consideration in turn leads us to propose anisotropy in the attractive oligomer interactions as

cause for the transition from ordered CF polymerization to precipitation Figure adapted with

permission from Miti et al Biomacromolecules 2015 16 (1) pp 326ndash335 figure 6

92

A2 Loss of Colloidal Stability Predicts Onset of Oligomer Precipitation

We attempted to replicate the precipitation boundary using another colloidal model We

observed two distinct modes of supramolecular assembly above the COC CF polymerization

and oligomeric precipitation (PPt) Given the oligomeric structure of precipitates this suggests

that monomers must first coalesce into oligomers before they assemble into CFs or O-Ppt (Fig

A11 b) As with RFs vs oligomers above the thermodynamically favorable CFs are kinetically

disfavored due to their prolonged lag time while the O-Ppts appear immediately The simplest

model for the rapid appearance of precipitates would be a diffusion limited colloidal instability

of the oligomers In diffusion limited precipitation the resulting structures have a branched linear

morphology with a fractal dimension of 18 47

However the Debye length at the salt

concentrations where precipitates are observed is just a few angstroms As a result CFs and

branched linear precipitates become energetically indistinguishable on the length scale of

electrostatic interactions Hence the difference between these states must originate instead from

the non-electrostatic binding interactions Assuming anisotropic binding interactions where the

oligomers are strongly interacting in the axial direction and weakly interacting along their radial

direction could explain the reduced solubility of the CFs relative to the precipitates and their

large persistence length relative to the Debye length We recently postulated similar anisotropy

of oligomer interactions in order to explain the prion-like self-replication of lysozyme oligomers

under physiological solution conditions Structural support for the polar vs radial anisotropy of

amyloid oligomer interactions is provided by the cylindrin structure reported for amyloid

oligomers formed by model peptides

As with the oligomer phase boundary we model the precipitation boundary as a quasi-

equilibrium between two states The first state is a dilute solution of oligomers If the

93

concentration of oligomers is cn then the chemical potential in the solution phase is ln(cn) The

second phase is the precipitate The chemical potential in this state arises primarily from the

energetics forming favorable oligomer-oligomer contacts and repulsive electrostatic interactions

ie

Here Frad is the free energy contribution from radial interactions

and

is that due to electrostatic interactions Since the timescales for precipitate formation

preclude significant annealing we assume that highly ordered polar contacts will be greatly

suppressed compared to the weaker radial contacts For this reason we are unable to extract a

value for the polar binding energy The polar binding energy determines the oligomer

concentration required for the formation of CFs which our experiments are unable to resolve

from the oligomer formation boundary

There will also be an entropic contribution to the precipitate chemical potential due to the

disordered nature of the precipitate However this contribution is a function of the precipitate

density which we do not expect to vary over the salt concentrations of interest because of the

short screening length Therefore the entropic contribution can be absorbed into the unknown

parameter Frad As a crude model we assume that precipitates will appear when the chemical

potential of the solution is greater than that of the precipitate Therefore the condition for the

onset of precipitation is

n

(4)

Since precipitation occurs when most of the protein is in the oligomer phase ie we

find that precipitation occurs for

(5)

94

where is a parameter that will be obtained by fitting Since the electrostatic energies

of linear assemblies and precipitates are essentially indistinguishable we used a cylinder

geometry to calculate the electrostatic energy contributions to the free energy per molecule 39

The resulting precipitation boundary is superimposed onto the experimental data in figure 4

Again the theoretical prediction closely follows the experimental results

Our expressions for the COC and precipitation boundary contain two free parameters A and B

which are related to the non-electrostatic contributions to the oligomerization and precipitation

free energies Since the electrostatic contributions are entirely repulsive these terms will be

dominated by cohesive forces like H-bonds or hydrophobic interactions Upon fitting Eqn 3 to

the experimentally determined COC we find A=4310-8

corresponding to a non-electrostatic

binding energy of -117 kT This is partially offset by an electrostatic repulsion that ranges from

84 kT to 37 kT as the salt concentration increases from 100 mM to 800 mM Therefore the net

binding energy per protein varies between 4kT and 10 kT For the precipitation fitting parameter

in Eqn 5 we find B = 2010-6

which corresponds to Frad = -152 kT With electrostatic

repulsion included the net oligomer-oligomer binding energy varies from 89 kT (400mM salt) to

114kT (800mM) This is comparable to the binding energy per monomer in the oligomer

Oligomer-oligomer contacts in the precipitate most likely involve multiple monomers interacting

by less attractive surfaces

95

Appendix B Methods Theoretical Background

In this section of the appendix I give a detailed description of the physical principles

behind the methods I used during my PhD work

B1 Static and Dynamic Light Scattering Theoretical Background

Interaction of light with matter yields an abundant quantity of information regarding the

structure and dynamics of a given system Scattering absorption and emission of

electromagnetic waves by matter are the central physical phenomena studied in order to obtain

information regarding a system of interest The temporal and spatial resolution of obtained

information depends on both the physical state of the system and the physical parameters of the

electromagnetic waves interacting with it3 Light scattering from small particles is omnipresent

natural phenomena that is responsible for many visual effects like the blue color of the sky on a

good day4 or the visualization of salt dissolving in water

5 In the field of soft condensed mater

light scattering is of particular value due to its ability to give detailed information about the

structure and nature of interactions of nanomesoscopic particles in aqueous solutions In the case

of protein aggregation studies light scattering methods reveal details about proteinslsquo molecular

weight in a monomeric state oligomeraggregate formation6 as well as protein aggregation

kinetics7

Light scattering measurements of proteins are ensemble measurements that rely on

analysis of scattered lightlsquos intensity by the protein solutions There are mainly two types of

information obtained from light scattering measurements on protein solutions time averaged

intensity based information (or otherwise known as static light scattering SLS) and time

dependent intensity fluctuations based information (also known as dynamic light scattering

DLS) represented in figure 1

96

Figure B1 Static Light

Scattering vs Dynamic Light

Scattering SLS is based on

intensity time average over ―long

time intervals (seconds) while

DLS is based on short interval

(milliseconds)intensity

fluctuations

Static light scattering provides information about particles molecular weight twomulti body

interactions between the particles in solutions and form factor of large particles (the former is

valid if a multi-angle set up is used) As an electric field interacts with a given molecule it

dislocates the moleculelsquos electrons towards one end of it thus the electric field induces a dipole

by polarizing the molecule When this induced dipole relaxes it emits the energy in form of a

photon having the same wavelength (elastic scattering) This way the physical picture of static

light scattering is based on matter reacting to electromagnetic waves by polarizing itself

Polarizability represents the measure of electronslsquo shift within a given molecule in the direction

of the applied field The induced dipolelsquos moment is directly proportional to both the applied

field and the polarizability of the given molecule8

=

Where permittivity of vacuum is is polarizability tensor and is the applied electric field

In the case of laser light scattering is the electric field associated with the electromagnetic

radiation hence it has a defined polarization Moreover for Rayleigh regime of scattering the

particles in solution are much smaller than the wavelength of the incoming electric field (about

λ20) hence the polarizability can be considered constant For particles in aqueous solution the

97

solvent has a well-defined polarizability (also constant) hence the light scattering is given by the

difference in polarizability of the solvent and the solute otherwise known as excess

polarizability

= (

Because the particles are much smaller than the wavelength of the electric field each particle

experiences a uniform oscillating electric field which induces at its turn an oscillating dipole

This oscillating dipole acts like an emitter of an isotropic electric field having the same

wavelength as the incoming electric field but slightly shifted phase This phase difference can be

neglected in Rayleigh regime for static light scattering

=

Where ω is 2πcλ is the angular frequency of light with wavelength λ is the wave vector

which indicates the direction the electromagnetic fieldlsquos direction of propagation | |=2πλ

and is the position of interest and is the phase difference which is neglected Hence the

time varying dipole moment becomes

(t) = ( =

The scattered electric field of an oscillating dipole electric field is then given by

= (

)

=-

where R = | | is the distance from scattering volume to the detector Usually the scattering

intensity is measured =

=

98

For most SLSDLS instruments electric field is vertically polarized (z-axis) while the detector

lays in the x-y plane meaning that θ is 0 Also the scattered electric filed is independent of φ

since it is isotropic in the Rayleigh regime

Experimentally the refractive index is easier to measure than polarizability it is easier to

express using9

hence

=

and

=

Where N is number density of particles in solution M is the molecular weight of the particles C

is the mass density of the particles NA is Avogadrolsquos number ε0 is the permittivity of vacuum

is the refractive index of the scattering particles and is the refractive index of solvent In

case the density fluctuations of solvent can be neglected it is easy to see that the scattering

intensity depends on the particleslsquo scattering power mass concentration and osmotic pressure

Where T is temperature is Boltzmanlsquos constant and is osmotic pressure From the

equation above is the scattering power for a particle and also known as optical contrast factor

K

K =

=

while osmotic pressure derived based on fluctuation theory developed by Smoluchowski and

Eistein10

99

=

for ideal solution where there is no inter-particle interactions

=

2 for non-deal solutions with Bn being the virial

coefficients

Scattering intensity also depends on the geometry of the SLSDLS apparatus The wavelength of

the laser used the distance between the detector the scattering sample and the scattering volume

are the main parameters that affect the scattering intensity Introducing the Rayleigh ratio is a

natural way of accounting for the experimental set-up as well as for any angular dependencies

that have been neglected based on particles size assumption For a multiangle set-up this ratio

has to be measured and calculated for every angle

= ( )

= ( )

Where and are the scattering of a standard and the normalized tabulated scattering

of the standard (usually toluene) Combining together the expressions for scattering intensity and

Rayleigh ratio it is possible to get an expression that can be directly used in the laboratory to

extract molecular weight and the virial coefficient that describes the two-body interactions of

particles in solution

=

+ 2 C +hellip

100

Figure B2 Static Light Scattering and Dynamic Light Scattering Experimental Diagram

Light from a laser passes through a solution and the scattered lightlsquos intensity is measured at

different angles in the plane perpendicular to the initial lightlsquos polarization A computer equipped

is calculating the correlation function in real time

Dynamic light scattering is mainly used to obtain particles diffusion coefficients as well as

correlation relaxation times in some systems like protein gels

Given that in solutions particles are subject to Brownian motion it is possible to measure

indirectly the particles sizes in solution through the diffusion coefficient The diffusion

coefficient measured in DLS experiments is linked to the hydrodynamic radius through Stokes

equation of a diffusing sphere with radius in a solution with viscosity η at temperature T11

D

The hydrodynamic radius is related to the measured diffusion coefficient by the Stokes-Einstein

equation

=

101

In reality the Stokes-Einstein has a general form that is dependent on protein concentration in

solution and its generalized form is given by

=

Where D is the diffusion coefficient M is the molecular weight f is the friction coefficient C is

the mass density of the particles NA is Avogadrolsquos number and is osmotic pressure Here the

expression becomes more complicated since both the diffusion coefficient and the friction

coefficient are concentration dependent

1 )

1

where the superscript ―0 indicates the values at zero protein concentration So together with the

expansion for the osmotic pressure given previously and the fact that 2

and that by definition =

the diffusion coefficient becomes

=

= 1 2

As it can be seen from the equation above in order to obtain one must perform DLS

measurements for a series of concentrations and then extrapolate to zero concentration It is easy

to see that the interaction parameter is obtained from static light scattering and is then used to

calculate and obtain the real diffusion coefficient

102

B2 Thiovlavin T Fluorescence Theoretical Background

In 1959 Vassar and Culling showed that the benzathiole derived dyes Thioflavin T and S

(ThTThS) have the greatest affinity for amyloid plaques and smaller number of false positives

out of a large set of tested dyes including Congo Red12

Later ThT was used to characterize

amyloid fibrils purified from patients and it was reported that the dye undergoes a red shift in its

excitation spectrum upon binding to amyloid fibrils from excitation and emission of 350 nm and

438 nm respectively to 450 nm and 482 nm and this shift was used to establish a linear

relationship between the amount of amyloid fibril and ThT fluorescence for the first time13ndash15

Next ThT was reported to be a great tool for monitoring amyloid fibril aggregation in vitro and

it became a valuable tool for monitoring amyloid fibril aggregation kinetics under a myriad of

protein solution conditions1617

Despite its wide use for monitoring amyloid fibrils aggregation the mechanism and modes of

ThT binding to the fibrils are still debated1618ndash24

Early experimental works characterizing ThT

spectral properties and first principle calculations have shown that ThT is a molecular rotor

displaying complex fluorescent behavior25ndash29

In a series of publications Stiapura and his

colleagues have shown that the electronic ground state of ThT S0 corresponds to a 37ᵒ

angle

between the C6 ndashC12 bond between the benzthiazole ring and benzene ring (φ in fig 1a) Upon

excitation the first locally exited state (LE) starts at φ = 37ᵒ and relaxes to the minimum of the

ThT exited singlet state S1 with the dihedral angle of φ = 90 through internal charge-transfer

(ICT) Upon spatial hindrance both through high solvent viscosity or binding to rigid structures

the nonradiative twisted internal charge-transfer (TICT) state is obstructed and the molecule

remains in the radiative locally exited state with φ close to 37ᵒ and relaxes through emissive

radiation 262830

103

ThT binding to amyloid fibrils is still an active area of research and it has been shown that its

binding modes are subject to specific fibril structure being mainly dictated by the steric and

coulomb interactions with the amino acid residues of the fibril core18ndash2331

The common theme

emerging from the studies on ThT binding to amyloid fibrils is the ThT binding along the axis of

the amyloid fibrils (its short axis perpendicular to the cross-β sheetlsquo strands) in the

grooveschannels created by amino acids residues of at least 5 β-strands18ndash202431

(fig 1b)

(a)

(b)

Figure B3 Thioflavin T Molecule (a) chemical structure and molecular sizes26

Notice the two

angles φ and ψ around the single bond that links the rigid structures of the molecule Rotations

around angle φ in the exited state correspond to either high or low quantum yield of ThT while

ψ anglelsquos rotations have minimal effect (b) My artistic impression Thioflavin T molecule bound

to the amyloid fibril core (based on text from references18ndash20

) Part (a) of this figure is adapted

with permission from Stiapura et al Journal of Physical Chemistry A 2007 111 pages 4829-

4835 figure 1

There are various reports on ThT binding to amyloid fibrils either as micelle dimer or individual

molecules21ndash233233

Recent works have shown that ThT binds in non-aggregated monomeric

form to HewL amyloid fibrils through a simple bimolecular mechanism2334

Also by means of

microdialysis Sulatskaya and her colleagues have identified that that there are two binding

104

modes of ThT to HewL amyloid fibrils at pH 2 Kb1 = 75 10-6

M-1

and Kb2 = 5610-4

M-1

with

different number density of dye binding sites on the amyloid fibrils n1 = 011 and n2 = 024

respectively2

B3 Protein Fourier Transform Infrared Spectroscopy Theoretical Background

Mid infrared radiation absorption excites vibrational and rotational transitions of

molecules Vibrational transitions are forbidden unless there are also molecular dipole moment

changes hence the strength (type) and the polarity of the intramolecular bonds as well as

coupling with other extramolecular effects have a strong influence on the absorption spectrum of

a given molecule36ndash40

With the introduction of Fourier transform infrared spectroscopy (FTIR)

and especially attenuated total reflectance technique (ATR) for water based solvents infrared

spectroscopy became a valuable structural probe for protein research40ndash42

Vibrations of peptide

bonds implicated protein secondary structure motifs give rise to nine IR bands with the Amide I

band yielding the most reliable and easy to analyze data3842ndash44

The frequencies constituting

amide I band (1700 ndash 1600 cm-1

) depend on stretching of C=O bonds and N-H of peptide bonds

(~80 and ~20 of its intensity respectively) and carry information about their

microenvironments hence the secondary structure to which they belong36ndash394546

105

(a)

(b)

Amide I Band Vibrational Modes

Contributions

Figure B4 Near FTIR Spectrum and Main Vibrational Modes Contributing to IR Bands

(a) FTIR spectra of biopolymers Notice the Amide I band and its regions in the insert47

(b)

vibrations modes responsible for amide I absorbance band C=O stretching has the biggest

contribution and N-H stretching has the second highest contribution167168

In a series of three papers in 1976 Chirgadze and Nevskaya have calculated and confirmed in

experiments the peaks of the two main structural motifs α-helix and β-sheets (parallel and anti-

parallel) based on analysis of pure α-helix and β-sheets formed by polyaminoacids

Table B1 Amide I Band Assignment to Protein Secondary Structure Motifs Empirical vs




Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548

1689 - 1698 β-sheets 1690(anti-parallel strong)3839

106

Further additions and refinements from other groups described all secondary structural motifs

within structurally complex proteins and their respective bands have been assigned within the

amide I region A summary of these assigned bands is summarized in Table B135ndash3843ndash45

Recently FTIR has started to be implemented as structural probe for identifying the

intermolecular β-sheets of amyloid fibrilslsquo core cross β-sheets which is shifted from the proteinlsquos

native β-sheets49ndash55

Moreover it has been shown that despite being a low resolution structural

probe FTIR is able to discern between amyloid fibrilslsquo core cross β-sheets formed by the same

protein under various solution conditions5054ndash56

Table B2 Comparison of Native vs Amyloid Fibrils Core β-sheets Notice that amyloid

fibrils core β-sheets are emerge at lower frequencies than the native β-sheets for β2

microglobulin and human lysozyme and appear de novo for Aβ42 and α-synuclein also at low

wavenumbers

ProteinPeptide Aggregate Species Low Wavenumbers β-

sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557

Fibrils (pH 74) 162057

-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

A summary of most commonly studied amyloidogenic proteinspeptides comparison of native

and amyloid core β-sheets is summarized above in table B25557ndash62

B4 Atomic Force Microscopy Theoretical Background

Since its introduction by Binnig Quate and Gerber in 1986 Atomic Force Miscroscopy

(AFM) has been an invaluable tool for getting high resolution topological images of

nonconductive materials6364

The introduction of the flexible cantilever with optical heterodyne

detection of the cantilever vibrations converted AFM into a high resolution (sub 100 Å) high

quality single molecule technique65

In recent decades AFM has become a highly employed

method for studying mature amyloid fibril morphologies as well as their temporal evolution from

pre-fibrillar aggregates to end stages766ndash70

A general operation diagram of an AFM is given in figure 1a As one can notice there are three

main components in the construction of an AFM piezoelectric transducers force transducers and

feedback control loop The force transducers measure the force between the tip (figure B41b)

and the sample the piezoelectric transducers move the AFM tip over the sample the feedback

control loops the signal from the force transducer back in to the piezoelectric in order to keep a

constant force between the tip and the sample or a fixed deflection of the cantilever depending

on the AFM scanning mode64

Usually the force transducer in an AFM is a cantilever with

integrated tip (the probe) and an optical lever (see fig B41b) During a measurement a laser

beam is reflected by the back reflective surface of the cantilever into a four-segmented

(quadrupole) photodetector The reflected lightlsquos path changes when the tip at the end of the

cantilever interacts with the sample This change is detected by the photodetector and the sensed

108

force is used by the feedback controller to set the z peizolsquos position (fig B41) Force

transducers are conventionally made of amorphous lead barium titanate (PdBaTiO3) or lead

zirconate titanate (Pb[ZrxTi1-x]O3 or PZT) They are 50 ndash 300 microM long and 20 ndash 60 microM long

and 02 ndash 1 microM thick Modern commercial AFMs use piezoelectric materials that can undergo

changes as low as 01 nm per volt applied and force transducers are capable of measuring forces

lower than 10 pN For non-contact imaging more often the cantilevers are made of silicone

nitride (Si3N4) while the probe itself is made of silicone and are much stiffer than the cantilever

(a)

Figure B5 AFM Construction and

Working Principles (a) Block-

diagram of AFM operation based on

reference 62 text description Notice the

three main components of an AFM the

force transducer which is also the

cantilever the piezoelectric crystals

responsible for independent movement

in any of the xyz directions and the

feedback electronic controller (b)

Designs for tip‐scanning AFM with

optical lever sensors based reference 62

text descriptions Notice how the

movements of the tip due to its

interaction with the sample translate

into the movement of the cantilever

The laser light reflecting of the

reflective surface on the back of the

cantilever is detected by the

photodetector Any change in the

lightlsquos path due to movements of the

cantilever is registered by one of the

four poles of the detector and is

translated into a voltage applied to the

PZT in order to regulate the force

(distance) between the sample and the

probe

(b)

109

One of the main factorslimitations of AFM image quality is the geometry and the dimension of

the probe647172

The image taken (the measured samplelsquos profile) with AFM is an actual

convolution between the sample shape and the probelsquos geometry (figure B42a) This way while

the height in AFM images is given reliably the widths are distorted resulting in much wider xy

dimensions (fig B42b) If the geometry of the probe is given by the manufacturer or is

measured using standards (usually 100 nm spheres) the images can be deconvoluted and the real

widths of imaged samples can be determined76472

(a)

(b)

Figure B6 Image Distortions Introduces by the AFM Tip (a) Effect of the geometry and

size of the AFM probe on the measured profile72

Notice how the diameter of the tip itself as well

as the tiplsquos end affect the samplelsquos measured profile (b) Distortion of a spherical bead imaged

with a circular tip71

Notice that the apparent width of a sample would be twice its actual value

even if the tiplsquos end diameter is only half the diameter of the measured sample itself Part (a) of

this picture is adapted with permission form Marinello at al J Manuf Sci Eng 2010 132

030903 figure 4 Part (b) of this picture is adapted from

httpexperimentationlabberkeleyedusitesdefaultfilesAFMImagesLecture_10_AFMpdf

110

B5 Circular Dichroism Spectroscopy Theoretical Background

Circular dichroism spectroscopy (CD) is a great tool for probing proteins secondary and

tertiary structure7374

This method is based on absorbance by a molecule of preferably left or

right polarized light As a result of preferably absorbing clockwise or counter clockwise rotating

light at a specific wavelength the addition of electric filed vectors after passing though the

sample traces an ellipse rather than a circle This deviation from a perfect circularly polarized

light is called ellipticity and is measured in degrees (in biomolecules is of order of

milidegrees)73

Figure B7 Far-UV CD spectra of

the main secondary structures motifs

in a protein Notice the α- helix

negative peaks at 208 nm and 222 nm

and positive at 193 nm the β-sheets

negative peak at 218 nm and positive

peak at 195 nm low ellipticity at 210

nm and the negative peak at 195 nm of

disordered motif (coil) adapted from

httpwwwfbsleedsacukfacilitiescd

Molecules that possess an intrinsic chirality or are placed in a chiral surrounding would manifest

circular dichroism In a protein peptide bond is the main absorber hence its environment would

modify the extent and type of polarized light it absorbs making it an excellent probe for

identifying the presence and (to some extent) percent of different secondary structure motifs73ndash75

Notice the α- helix negative peaks at 208 nm and 222 nm and positive at 193 nm the β-sheets

negative peak at 218 nm and positive peak at 195 nm low ellipticity at 210 nm and the negative

111

peak at 195 nm of disordered motif (coil) Spectrum of a complex protein is a linear combination

of the spectra for individual structural motifs in proportion to their presence in the protein

B6 Gel Electrophoresis Theoretical Background

Gel electrophoresis is a great method of separating biopolymers based on their molecular

weight Since its introduction by Smithies in 1955 it became a central method for separating and

identifying proteins complexes into components as well as identifying any changes to them that

would affect their molecular weight including chemical or aggregation585976ndash79

Gel electrophoresis is based on migration of electrically charged biopolymers embedded in a

(usually) polyacrylamide matrix under the influence of an electric field76

The matrix pores

determine the maximum molecular weight that can migrate as well as the resolution of the

separation into individual bands the higher the concentration of polyacrylamide the smaller the

pores within the matrix hence smaller molecular weights can migrate and the resolution is

better79

Also for increased resolution additives that further decrease pore sizes (ex ammonium

persulfate) are added during the matrix preparation and those that help achieving band better

resolution are added in the running buffer77

In SDS-Page electrophoresis proteins are first

thermally denatured and then mixed with sodium dodecyl sulfate in order to charge uniformly all

proteins so that the migration in the electric field would be solely based on their molecular

weight β-mercaptoethanol is usually used to disrupt any disulfide bonds within the protein

112

Appendix C Copyright Permissions

In this part of the appendix I have pasted the print screens of the emails or websites of

the journals from witch I copied reproduced or partially reproduced some of the figures and text

(see figure captions)

Structural fingerprints and their evolution during oligomeric vs oligomer-free amyloid

fibril growth

Joseph Foley1 a)

Shannon E Hill1 a)b)

Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1

The Journal of Chemical Physics 139 121901 (2013) httpsdoiorg10106314811343

113

Stable Metastable and Kinetically Trapped Amyloid Aggregate Phases

Tatiana Mitidagger Mentor Mulajdagger Jeremy D SchmitDagger and Martin Muscholdagger

dagger Department of Physics University of South Florida Tampa Florida 33620 United States

Dagger Department of Physics Kansas State University Manhattan Kansas 66506 United States

Biomacromolecules 2015 16 (1) pp 326ndash335

DOI 101021bm501521r

Publication Date (Web) December 3 2014

Copyright copy 2014 American Chemical Society

E-mail mmuscholusfedu Phone (813) 974-2564

ACS AuthorChoice - This is an open access article published under an ACS

AuthorChoice License which permits copying and redistribution of the article or any adaptations

for non-commercial purposes

114

Permission from the Journal of Physical Chemistry Chemical Physics to reproduce figures from

reference 44 (P Arosio T P J Knowles and S Linse Phys Chem Chem Phys 2015 17

7606 DOI 101039C4CP05563B)

115

Life is an open access journal so I can use figure 1 from Smith JE Mowles AK Mehta

AK Lynn DG Looked at Life from Both Sides Now Life 2014 4 887-902

116

117

118

119

Marinello at al J Manuf Sci Eng 2010 132 030903 figure 4

120

APENDIX REFERENCES

1 Gill S C amp von Hippel P H Calculation of Protein Extinction Coefficients from Amino

Acid Sequance Data Anal Biochem 182 319ndash326 (1989)


Amyloid Fibrils Stoichiometry and Affinity of Dye Binding Absorption Spectra of

Bound Dye J Phys Chem B 115 11519ndash11524 (2011)

3 Mishchenko M I Travis L D amp Lacis A A Scattering Absorption and Emission of

Light by Samll Particles 1 (CAMBRIDGE UNIVERSITY PRESS 2002)

4 Smith G S Human color vision and the unsaturated blue color of the daytime sky Am J

Phys 73 590 (2005)

5 Benvenuto M A Gelatin and the Tyndall Effect A Colorful and Tasty Demonstration

Chem Educ 6 95ndash96

6 Harding S E amp Jumel K Light Scattering Curr Protoc Protein Sci 781-7814 (1998)



8 Oslashgendal L Light Scattering a Brief Introduction Univ Cph (2013)

9 Schaumlrtl W Light Scattering from Polymer Solutions and Nanoparticle Dispersions

(Springer Berlin Heidelberg 2007)

10 Johnson C H amp Gabriel D A Laser Light Scattering (Dover Publications 1995)

121

11 Scott D J Harding S E amp Winzor D J Concentration dependence of translational

diffusion coefficients for globular proteins The Analyst 139 6242ndash6248 (2014)

12 Vassar P S amp Culling C F Fluorescent stains with special reference to amyloid and

connective tissues Arch Pathol 68 487ndash498 (1959)

13 Naiki H Higuchi K Hosokawa M amp Takeda T Fluorometric determination of amyloid

fibrils in vitro using the fluorescent dye thioflavin T1 Anal Biochem 177 244ndash249 (1989)

14 Naiki H et al Fluorometric Examination of Tissue Amyloid Fibrils in Murine Senile

Amyloidosis Use of the Fluorescent Indicator Thioflavine T 393ndash396 (1991)

doi101007978-94-011-3284-8_99

15 Kuznetsova I M Sulatskaya A I Uversky V N amp Turoverov K K Analyzing

Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

PLoS ONE 7 e30724 (2012)

16 Naiki H Higuchi K Nakakuki K amp Takeda T Kinetic analysis of amyloid fibril

polymerization in vitro J Tech Methods Pathol 65 104ndash110 (1991)

17 LeVine H III Stopped-Flow Kinetics Reveal Multiple Phases of ThioflavinT Binding to

Alzheimer Beta(1-40) Amyloid Fibrils Arch Biochem Biophys 342 306ndash316 (1997)





(2009)

20 Krebs M R H Bromley E H C amp Donald A M The binding of thioflavin-T to amyloid

fibrils localisation and implications J Struct Biol 149 30ndash37 (2005)

122


229ndash238 (2005)



23 Ivancic V A Ekanayake O amp Lazo N D Binding Modes of Thioflavin T on the Surface

of Amyloid Fibrils Studied by NMR ChemPhysChem 17 2461ndash2464 (2016)

24 Kitts C C amp Bout D A V Near-Field Scanning Optical Microscopy Measurements of

Fluorescent Molecular Probes Binding to Insulin Amyloid Fibrils J Phys Chem B 113

12090ndash12095 (2009)

25 Voropai E S et al Spectral properties of thioflavin T and its complexes with amyloid

fibrils J Appl Spectrosc 70 868ndash874 (2003)

26 Stsiapura V I Maskevich A A Kuzmitsky V A Turoverov K K amp Kuznetsova I M

Computational Study of Thioflavin T Torsional Relaxation in the Excited State J Phys

Chem A 111 4829ndash4835 (2007)

27 Maskevich A A et al Spectral Properties of Thioflavin T in Solvents with Different

Dielectric Properties and in a Fibril-Incorporated Form J Proteome Res 6 1392ndash1401

(2007)

28 Stsiapura V I Maskevich A A Tikhomirov S A amp Buganov O V Charge Transfer

Process Determines Ultrafast Excited State Deactivation of Thioflavin T in Low-Viscosity

Solvents J Phys Chem A 114 8345ndash8350 (2010)

29 Amdursky N Erez Y amp Huppert D Molecular Rotors What Lies Behind the High

Sensitivity of the Thioflavin-T Fluorescent Marker Acc Chem Res 45 1548ndash1557 (2012)

123

30 Sulatskaya A I Maskevich A A Kuznetsova I M Uversky V N amp Turoverov K K

Fluorescence Quantum Yield of Thioflavin T in Rigid Isotropic Solution and Incorporated

into the Amyloid Fibrils PLoS ONE 5 e15385 (2010)

31 Robbins K J Liu G Selmani V amp Lazo N D Conformational Analysis of Thioflavin T

Bound to the Surface of Amyloid Fibrils Langmuir 28 16490ndash16495 (2012)

32 Groenning M et al Binding mode of Thioflavin T in insulin amyloid fibrils J Struct Biol

159 483ndash497 (2007)

33 Reinke A A amp Gestwicki J E Insight into Amyloid Structure Using Chemical Probes

Amyloid Structure Using Chemical Probes Chem Biol Drug Des 77 399ndash411 (2011)

34 Qin Z et al Kinetic Mechanism of Thioflavin T Binding onto the Amyloid Fibril of Hen

Egg White Lysozyme Langmuir Web Publication (2017)

35 Foley J et al Structural fingerprints and their evolution during oligomeric vs oligomer-free

amyloid fibril growth J Chem Phys 139 121901 (2013)

36 Chirgadze Y N amp Nevskaya N A Infrared spectra and resonance interaction of amide-I

vibration of the antiparallel-chain pleated sheet Biopolymers 15 607ndash625 (1976)

37 Nevskaya N A amp Chirgadze Y N Infrared spectra and resonance interactions of amide-I

and II vibrations of α-helix Biopolymers 15 637ndash648 (1976)



39 Bandekar J amp Krimm S Normal mode spectrum of the parallel-chain β-sheet Biopolymers

27 909ndash921 (1988)

40 Barth A Infrared spectroscopy of proteins Biochim Biophys Acta BBA - Bioenerg 1767

1073ndash1101 (2007)

124

41 Singh B R in Infrared Analysis of Peptides and Proteins (ed Singh B R) 750 2ndash37

(American Chemical Society 1999)

42 Byler D M amp Susi H Examination of the secondary structure of proteins by deconvolved

FTIR spectra Biopolymers 25 469ndash487 (1986)

43 Kong J amp Yu S Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary

Structures Acta Biochim Biophys Sin 39 549ndash559 (2007)

44 Besley N A Computing protein infrared spectroscopy with quantum chemistry Philos

Trans R Soc Math Phys Eng Sci 365 2799ndash2812 (2007)

45 Kumosinski T F amp Unruh J J Quantitation of the global secondary structure of globular

proteins by FTIR spectroscopy comparison with X-ray crystallographic structure Talanta

43 199ndash219 (1996)


vibration of the parallel-chain pleated sheet Biopolymers 15 627ndash636 (1976)



(2013)





3314ndash3321 (2009)

125

50 Sarroukh R Goormaghtigh E Ruysschaert J-M amp Raussens V ATR-FTIR A

rejuvenatedlsquo tool to investigate amyloid proteins Biochim Biophys Acta BBA - Biomembr

1828 2328ndash2338 (2013)

51 BOUCHARD M ZURDO J NETTLETON E J DOBSON C M amp ROBINSON C V

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron

microscopy Protein Sci 9 1960ndash1967 (2000)

52 Benseny-Cases N Coacutecera M amp Cladera J Conversion of non-fibrillar β-sheet oligomers

into amyloid fibrils in Alzheimerlsquos disease amyloid peptide aggregation Biochem Biophys

Res Commun 361 916ndash921 (2007)

53 Faumlndrich M On the structural definition of amyloid fibrils and other polypeptide aggregates

Cell Mol Life Sci 64 2066ndash2078 (2007)



55 Mossuto M F et al The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence

Cytotoxicity J Mol Biol 402 783ndash796 (2010)



57 Cerf E et al Antiparallel β-sheet a signature structure of the oligomeric amyloid β-peptide

Biochem J 421 415ndash423 (2009)



59 Chen S W et al Structural characterization of toxic oligomers that are kinetically trapped

during α-synuclein fibril formation Proc Natl Acad Sci 112 E1994ndashE2003 (2015)

126

60 Fabian H et al Early Stages of Misfolding and Association of β2-Microglobulin Insights

from Infrared Spectroscopy and Dynamic Light Scattering Biochemistry (Mosc) 47 6895ndash

6906 (2008)





63 Binnig G Quate C F amp Gerber C Atomic force microscope Phys Rev Lett 56 930

(1986)

64 Eaton P amp West P Atomic Force Microscopy (Oxford University Press 2010)

doi101093acprofoso97801995704540010001

65 Martin Y Williams C C amp Wickramasinghe H K Atomic force microscope-force

mapping and profiling on a sub 100-Aring scale J Appl Phys 61 4723ndash4729 (1987)

66 Drolle E Hane F Lee B amp Leonenko Z Atomic force microscopy to study molecular

mechanisms of amyloid fibril formation and toxicity in Alzheimerlsquos disease Drug Metab

Rev 46 207ndash223 (2014)





69 Flamia R Salvi A M DlsquoAlessio L Castle J E amp Tamburro A M Transformation of

Amyloid-like Fibers Formed from an Elastin-Based Biopolymer into a Hydrogel An X-ray

127

Photoelectron Spectroscopy and Atomic Force Microscopy Study Biomacromolecules 8

128ndash138 (2007)

70 Adamcik J amp Mezzenga R Study of amyloid fibrils via atomic force microscopy Curr

Opin Colloid Interface Sci 17 369ndash376 (2012)

71 httpexperimentationlabberkeleyedusitesdefaultfilesAFMImagesLecture_10_AFMpdf

Lecture_10_AFM

72 Marinello F Carmignato S Voltan A Savio E amp Chiffre L D Error Sources in

Atomic Force Microscopy for Dimensional Measurements Taxonomy and Modeling J

Manuf Sci Eng 132 030903 (2010)



74 Greenfield N J Using circular dichroism spectra to estimate protein secondary structure

Nat Protoc 1 2876ndash2890 (2007)

75 Tamburro A M Lorusso M Ibris N Pepe A amp Bochicchio B Investigating by circular

dichroism some amyloidogenic elastin-derived polypeptides Chirality 22 E56ndashE66 (2010)

76 Smithies O Zone electrophoresis in starch gels group variations in the serum proteins of

normal human adults Biochem J 61 629 (1955)

77 Schaumlgger H amp Von Jagow G Tricine-sodium dodecyl sulfate-polyacrylamide gel

electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem

166 368ndash379 (1987)



128

79 Schagger H Link T A Engel W D amp von Jagow G Isolation of the eleven protein

subunits of the bc1 complex from beef heart Methods Enzymol 126 224ndash237 (1986)


Scholar Commons

November 2017


Tatiana Miti

Scholar Commons Citation

Th esis


by

Tatiana Miti

A dissertation submitted in partial fulfillment

of the requirements for a degree of

Doctor of Philosophy

Department of Physics

College of Arts and Sciences


Major Professor Martin Muschol PhD

Vladimir Uversky PhD

Sagar Pandit PhD

Ghanim Ullah PhD

Robert Hoy PhD

Piyush Koria PhD

Date of Approval

June 27 2017

Keywords Phase Diagram Nucleated Polymerization Nucleated Conformational Conversion

Copyright copy 2017 Tatiana Miti

ACKNOWLEDGMENTS

















biophysics






training me










presence





adversity




i

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vi

I Introduction 1


























IV Results 32


Morphologies 32

ii














Spectroscopy 51












Appendices 89









Background 104






iii

LIST OF TABLES







iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis

ACKNOWLEDGMENTS

















biophysics






training me










presence





adversity




i

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vi

I Introduction 1


























IV Results 32


Morphologies 32

ii














Spectroscopy 51












Appendices 89









Background 104






iii

LIST OF TABLES







iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis






training me










presence





adversity




i

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vi

I Introduction 1


























IV Results 32


Morphologies 32

ii














Spectroscopy 51












Appendices 89









Background 104






iii

LIST OF TABLES







iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis




i

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vi

I Introduction 1


























IV Results 32


Morphologies 32

ii














Spectroscopy 51












Appendices 89









Background 104






iii

LIST OF TABLES







iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis

ii














Spectroscopy 51












Appendices 89









Background 104






iii

LIST OF TABLES







iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis

iii

LIST OF TABLES







iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis

iv

LIST OF FIGURES






and pH 7 29



















v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis

v


















vi

ABSTRACT



















vii























1

I INTRODUCTION




















2







heavy chains

AL and AH



amyloidosis




Lysozyme ALys



Secondary systemic

amyloidosis



angiopathy

Cystatin C ACys


amyloidosis

Gelsolin AGe

Familial amyloid

polyneuropathy I


Familial amyloid

polyneuropathy II





3



literature














that time1116





4



























5




(a)

(b)

(c)

(d)

(e)

(f)




plateau43



(c) Morphologies of









6




Figure 22






sheets together3738



















7






Initial in vivo


populations4647


gathered 63ndash66








protein 6970










8





His





















9


























10








Lansbury48101102







So the











11



in






They used protein


















12





remained unanswered

















13















amyloid aggregates




14

















15









412120


cm-1











protocol)

16























17














accuracy improved






18












(a)


19

(b)












20














Formation Kinetics








21























22

aggregates35










and 4000 cm-1

All















to 1700 cmminus1

The


23



















Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 1663126

1667 - 1685 β-turns 1665127


strong)128129

24


and








β-sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



1695130

Fibrils (pH 74) 1620130

-


Oligomers (pH 74) 1625131132

1695131132

Fibrils (pH 74) 1628131132

-


-


1684133

Fibrils (pH 21) 1620133

-


-


1688134


1693135

Fibrils (pH 2) 1622135136

-

25






















26
















27





properties137









HewL124


HewL is a 129











28



(fig 3 )

(a)

(b)




29






(a)

(b)

(c)













30



Recently a new













amyloid core154







but its






31



(a)

(b)




aggregation154



32

IV RESULTS


Spectral Properties
















33









(a)

(b)



34

(c)

(d)




















35

(a)

(b)

(c)

(d)










36






Monomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)


()



()




Oligomeric Fibril

Assembly

Height

(nm)

Width

(nm)

Cross-section

(nm2)

Volume

(nm3)




Fibrils









37














(a)

(b)






38



















and the




39





158161



Despite the
























40

(a)



























2013 139(12)121901 figure 2


(b)

(c)



41




(a)

(b)

(c)




















42
























43

(a)

(b)

(c)

(d)









44




















Figure







if hydrolysis








45



)







into CF

(a)

(b)









46























47



(a)

(b)

(c)








48













figure 1














49

(a)

(b)



















50











overcome


Metastable










51




hours is RF





















52

(a)

(b)

(c)

(d)














53












Boundary

















54



(a)

(b)


















55






Phase Diagram





= ( - COC)COC






= ( - SRF) SRF




56



COC









(a)

(b)

(c)






57


























58



(a)

(b)

(aI)

(aII)

(bI)

(bII)

(bIII)

(bIV)

(bV)





59





























60
















At









61














two scenarios



(fig 14 fig 17b)

62
















63







(a)

(b)











64







The

















65







Concentration






or RF (orange)










66



















67













aggregates52










signature

68
























69
























70




71

REFERENCES


Med 337 898ndash909 (1997)



142ndash159 (2012)


12 (2016)


11 (2015)


1300 (2012)





1810 (1927)



72


(2000)



(2009)




229ndash238 (2005)









1150ndash1151 (1971)




231ndash252 (1971)

73





1068 (1985)



Commun 141 782ndash789 (1986)





175 1159ndash1164 (1991)



Commun 425 534ndash539 (2012)


(1982)





74


(1999)






J Mol Biol 273 729ndash739 (1997)







2375ndash2380 (2001)


(2005)




Nature 447 453ndash457 (2007)



75



(2002)


Biophys 39 1 (2006)






887ndash902 (2014)












76


10 S10ndashS17 (2004)





(2003)







3883 (2011)









77





2292 (2004)







440 352ndash357 (2006)





Biol Chem 289 35781ndash35794 (2014)







78












Phys 135 065107 (2011)



171 (2012)



Rev E 89 (2014)



1794 375ndash397 (2009)



79







(1952)




4864ndash4868 (1974)



(1980)




Chem 258 3207ndash14 (1983)



22 5836ndash5843 (1983)

80



(1983)



(1986)




10382ndash10400 (1997)








1913 (1982)



1044 (1967)



81












100 12069ndash12074 (2003)












82






















83



2071 (2011)



Soc 126 1992ndash2005 (2004)









1767 1073ndash1101 (2007)









84











(2015)



47 6895ndash6906 (2008)









85


1986)










(1969)











86








(2014)





FASEB J 26 192ndash202 (2012)









991 (2007)

87



3314ndash3321 (2009)



1668 1ndash27 (1996)










(2014)







632ndash643 (2015)

88



(2013)



89

APPENDICES


Theoretical Model










form an nth


(1)






power of the

90




(2)








(3)









D Finally the




91






(a)

(b)

(c)


















92

























93




ie


and














n

(4)



(5)

94





















95


















kinetics7







96








fluctuations











=






97



polarizability

= (






=





(t) = ( =


= (

)

=-



=

98





express using9

hence

=

and

=








K

K =

=


Eistein10

99

=


=


coefficients







= ( )

= ( )






=

+ 2 C +hellip

100











D


equation

=

101



=





1 )

1





=

= 1 2





102




















angle







radiation 262830

103




The common theme




(fig 1b)

(a)

(b)


Notice the two







4835 figure 1





Also by means of


104


M-1

and Kb2 = 5610-4

M-1

with


respectively2


















105

(a)

(b)


Contributions



(b)










Frequency (cm-1


Associated

Theoretical Value



1644 - 1654 Random -

1654 - 1657 α-helix 166337

1667 - 1685 β-turns 166548


106













wavenumbers


sheet Peak (cm-1

)

High Wavenumbers

β-sheet Peak (cm-1

)



169557


-



16955859

Fibrils (pH 74) 16285859

-


-


168460


-


-


168862


169355

Fibrils (pH 2) 1622355561

-

107

























108








(a)



























probe

(b)

109


the probe647172







(a)

(b)











110









milidegrees)73

















111










The matrix pores




better79








112






fibril growth

Joseph Foley1 a)


Tatiana Miti1 a)

Mentor Mulaj1 a)

Marissa Ciesla1


113






DOI 101021bm501521r







114



7606 DOI 101039C4CP05563B)

115



116

117

118

119


120

APENDIX REFERENCES









Phys 73 590 (2005)










121









doi101007978-94-011-3284-8_99



PLoS ONE 7 e30724 (2012)









(2009)



122


229ndash238 (2005)







12090ndash12095 (2009)





Chem A 111 4829ndash4835 (2007)



(2007)






123







159 483ndash497 (2007)














27 909ndash921 (1988)


1073ndash1101 (2007)

124











43 199ndash219 (1996)





(2013)





3314ndash3321 (2009)

125



1828 2328ndash2338 (2013)





















126



6906 (2008)






(1986)


doi101093acprofoso97801995704540010001





Rev 46 207ndash223 (2014)







127


128ndash138 (2007)




Lecture_10_AFM



Manuf Sci Eng 132 030903 (2010)











166 368ndash379 (1987)



128




Scholar Commons

November 2017


Tatiana Miti


Th esis

Thermodynamic and Kinetic Aspects of Hen Egg White ...

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Transcript of Thermodynamic and Kinetic Aspects of Hen Egg White ...