Regular class times: MWF 10-10:50 AM shapiro/biophysics12/ Instructors: (1) Professor Martin...

Regular class times: MWF 10-10:50 AM

http://www.wfu.edu/~shapiro/biophysics12/

Instructors: (1) Professor Martin Guthold, Phone: 758-4977, Office: 302 Olin, email:[email protected],

http://www.wfu.edu/~gutholdm/

(2) Professor Kim-Shapiro, Phone: 758-4993, Office: 208 Olin, email: [email protected], http://www.wfu.edu/~shapiro/

Office hours: Guthold: M, W, F; 1:30 pm – 2:30 pm, and by appointment. Kim-Shapiro: M, W; 2:15 pm -4:00 pm, and by appointment

Texts: 1. Principles of Physical Biochemistry, by K.E. van Holde, W. C. Johnson, and P.S. Ho2. Neurodynamix, by W.O. Friesen and J.A. Friesen.

3. Supplementary texts on reserve: 1. Biophysical Chemistry Part II, Techniques for the study of biological structure and

function, by Charles Cantor and Paul Schimmel (1980).2. Biochemistry by Lupert Stryer (1988). 3. Additional reading will be assigned in the form of journal articles and handouts

Physics 307/607 Biology 307/607

Physics 307/607 Biology 307/607Syllabus

Grading:

Undergraduate Students: 2 Midterm exams...........................40% Project………………………………10%Final Exam.....................................30 % Problem Sets..................................20%

Emphasis in grading will be placed on how each problem is solved. All work showing how the solution was obtained must be shown. An answer with the correct answer but poor method is inferior to one with the wrong answer but good method. Problem sets will generally be assigned for each chapter and the students will have one week to complete them. Students may help each other on problem sets but each student must write their own solution to each problem. The project that all students do will be a 5-10 page paper focusing on a particular topic in biophysics. The project could be a service learning project (see instructors for more information on that). Project topic is due in two weeks (Feb. 1)Project outline is due before spring break (March 9)Complete project due last day of class (May 2)

** Graduate students need to do a 5-10 minutes presentation on one of the journal articles that are part of the reading assignments (see reading list); or another article relevant to a lecture topic.

Graduate Students:2 Midterm exams........................... 30% Project……………………………….10%Presentation of Journal Article.......10% ** Final Exam.....................................30 % Problem Sets.................................20%

Exam Schedule:

Midterm 1: Monday, Feb. 27 (in-class)

Midterm 2: Friday, April 20 (in-class)

Final Exam: Monday, May 9, (9:00 am – 12:00 pm)

Miscellaneous:

We will, at times, look at structures that are deposited in the protein data bank (http://www.rcsb.org/pdb/home/home.do). The data bank contains the coordinates of all solved protein, DNA, RNA and other bio-molecular structures, usually to atomic resolution.


http://www.rcsb.org/pdb/home/home.do

Tentative Syllabus:

Part I Biophysical Methods

1. Introduction (Guthold) (~6 lectures)1.1 Biological Macromolecules; 1.2 Molecular interactions; 1.3 Overview of ThermodynamicsReading: van Holde, chapters 1-4 (partial).

2. X-ray diffraction, DNA Structure (Guthold) (~5 lectures)Fourier Transforms, Scattering, (x) F(q), A helix , History of Watson and Cricks' discovery and its

implications Reading: van Holde chapter 6, Watson and Crick Papers

3. Light Scattering, Sedimenation, Gel Electrophoresis, Higher Order DNA Structure (Kim-Shapiro) (~4 lectures)

Sedimenation, mass spectrometry, Gel electrophoresis (Fick's Law), Light Scattering (Classical, Dynamic, Polarized) DNA Topology (Length, Twist, and Writhe), Chromosome Structure Reading: van Holde, chapters 5 and 7, Polarized Light Scattering

4. Absorption Spectroscopy, Protein Structure (Kim-Shapiro) (~4 lectures)UV, VIS spectroscopy, linear and circular dichroism Protein primary, secondary, tertiary, quaternary structure Reading: van Holde chapters 8-10


Tentative Syllabus (cont.)

5. Emission Spectroscopy (Guthold) (~4 lectures)Reading: van Holde, Chapter 11

6. Single Molecule biophysics (Guthold) (~3 lectures)Reading: Chapter 16

7. Electron Paramagnetic Resonance, Protein Function - Hemolgobin (Kim-Shapiro) (~4 lectures)Electron Paramagnetic Resonance, Hemoglobin cooperativity Studies using EPR and time-resolved

absorption spectroscopy Reading: Handout

Part II Membrane Biophysics

8. Biological membranes and Transport (Kim-Shapiro) (~4 lectures)Description of membranes, Diffusion, Facilitated transport, Nernst Equation, Donnan EquilibriumReading: van Holde chapters 13-14

9. Nerve Excitation (Kim-Shapiro) (~3 lectures)Neurons, Action Potential, Propagation of action potential, measurements in membrane biophysics,Synaptic transmission Reading: Frisens Sections 1 and 2


Reading: Van Holde, Chapter 1

Van Holde Chapter 3.1 to 3.3

Van Holde Chapter 2

(we’ll go through Chapters 1 and 3 first.)

Homework (due Wednesday, Jan. 25):

1. What is the Central Dogma of Molecular Biology (describe, sketch in your own words)?

2. Van Holde 1.2 (amino acid structure)3. Van Holde 1.4 (amino acid structure)4. Van Holde 1.7 (DNA structure)5. Protein data bank exercises (see extra handout; protein & DNA structure)

Paper list (for presentations) is posted on web site

http://www.wfu.edu/~shapiro/biophysics12/

Introduction-1Structures of biological Macromolecules

Bovine pulmonary artery endothelial cell(image: Justin Sigley, WFU Physics)

• In this course we will mainly deal with:

proteins, nucleic acids, and membranes

• Physical methods to examine the structure and function of these biological molecules

From Voet & Voet Biochemistry


(e.g. DNA, RNA)

(e.g cell walls)


Outline

• Central Dogma, Replication, Transcription, Translation

• Genetic code, DNA/RNA codons

• Nucleic acids, DNA, RNA

• DNA structure, twist, rise, linking number

• Amino acids, proteins

• Protein structure, 1o,2o, 3o, 4o structure

• Properties of amino acids, (small, large, neutral, charged, hydrophobic, hydrophilic, etc.)

• Protein data bank (PDB)

Biological Macromolecules – General Prinicples

- Well-defined stoichiometry & geometry. Not readily broken into tiny pieces

- Monomer is the building block (amino acid→proteins, nucleic acid→DNA/RNA)

(Macro = large. Up to ~ 25 residues = oligomer; >25 polymer)

• 1° structure: one-dimensional sequence

• 2° structure: local arrangement (-helices, -sheets, turns)

→super secondary structures: hairpins, corners, motifs, etc.

• 3° structure: 3-D structure (e.g. folded protein), stabilized by H-bond, hydrophobic forces, van-der-Waals, charge-charge, etc

• 4° structure: Arrangement of subunits (e.g. hemoglobin)

- Configuration vs. Conformation:

• Configuration – Defined by chemical (covalent bonds), must break bond to change configuration (e.g. L-amino acid, D-amino acid)

• Conformation – Spatial arrangement (e.g. an amino acid polymer can have a huge number of different conformations, one of which is the natively folded protein).

Central dogma of Molecular Biology

Describes how the genetic information encoded (stored) in the ‘letter sequence’ of DNA is first transcribed and then translated into an amino acid sequence, i.e. into proteins. (Crick, F.H.C. (1958): On Protein Synthesis. Symp. Soc. Exp. Biol. XII, 139-163.Crick, F. H. C. (1970): Central Dogma of Molecular Biology. Nature 227, 561-563.)

(Enzymes catalyze reactions in organism)(Proteins – building blocks of organism)

The genome, or genomic DNA (deoxyribonucleic acids), of an organism consists of a very long sequence of four different nucleotides with bases A, C, G, T. Genomic DNA is a double-stranded helix comprised of two complementary strands, held together by A-T and C-G base pairs. The entire genome is replicated by DNA polymerases (a protein) and passed on to daughter cells during cell division. The genome consists of many (usually thousands) of genes. A gene is a specific, defined nucleic acid sequence that encodes one particular protein. The human genome consists of about 3·109 base pairs and only about 30,000 genes (in higher organisms, large parts the genome (80 – 98%) do not encode any known proteins).

Genomic DNA

Re

plic

atio

n

(DN

A p

oly

me

rase

)

mRNA

Transcription

(RNA polymerase)

Transcription: RNA polymerase (a protein) binds to the beginning of one particular gene and synthesizes an exact RNA copy of that gene. RNA (ribonucleic acid) consists of nucleotides with bases A, C, G, U. It is single-stranded. Transcription stops at the end of each gene and the RNA chain is released. A gene is on the order of a thousand bases.

Protein

Tran

slat

ion

(Rib

osom

e)

Translation: The RNA is moved to the ribosome. The ribosome reads the RNA sequence (with the help of t-RNA) and synthesizes an amino acid chain (polypeptide). The polypeptide folds into a three-dimensional structure – a protein (or part of a protein). There are 20 different amino acids, thus three RNA letters are needed to code for one amino acid. These triplets of RNA letters are called codons.

http://profiles.nlm.nih.gov/SC/B/B/F/T/_/scbbft.pdf

Central dogmaPicture in prokaryotic (bacterial) cell and eukaryotic (higher) cell

Eukaryotic cell

The human genome has about 30,000 genes (and lots of non-coding DNA)

Simply speaking: one gene one polypeptide

Prokaryotic cell (no nucleus)

Transcription (making RNA from a DNA template):

RNA polymerase binds at a promoter (beginning of a gene), unwinds DNA, and starts synthesis of an RNA copy of the gene

The sequence of bases in DNA codes for the sequence of amino acids in proteins

First real-time movies of a transcribing RNA polymerase 1,2

1. S. Kasas et al., Biochemistry 36, 461 (1997). (see Fig. 16.6 of book)2. M. Guthold et al., Biophysical Journal 77, 2284 (Oct, 1999). Kasas movie

http://www.youtube.com/watch?v=ZDH8sWiUsAM

http://www.youtube.com/watch?v=YEzRz1jmqNACredit: 8 minute movie of inner workings of a cellBioVisions, Harvard University

http://www.youtube.com/watch?v=YEzRz1jmqNA

http://www.rit.edu/~gtfsbi/IntroBiol/images/CH09/figure-09-07.jpg

Nucleosome

How to compact 2 meters of DNA into 2 m-sized nucleus?(like folding a 1000 km long long fishing line (1 mm diameter) into 1m sized ball)

The structure of DNA and RNA

• Four monomer building blocks

• RNA has ribose instead of 2’-deoxyribose

• RNA has Uridine instead of Thymidine

Stabilizing factors in double-stranded (ds)-DNA

This is also how DNA and RNA match up (hybridize) in the binding pocket of RNA polymerase during transcription!!

Normal Watson-Crick base pairing

Base Base plus ribose sugar Nucleoside (RNA)

Base plus deoxy ribose sugarDeoxy-nucleoside (DNA)

Base plus ribose sugar plus phospate (nucleotide)*

Adenine (A) Adenosine (A) Deoxy-adenosine (dA) Adenosine monophospate (AMP)

Cytosine (C) Cytidine (C) Deoxy-cytidine (dC) Cytidine monophospate (CMP)

Guanine (G) Guanosine (G) Deoxy-guanosine (dG) Guanosine monophospate (GMP)

Thymine (T) (Methyluridine, m5U) Thymidine (dT) m5UMP

Uracil (U) Uridine (U) Deoxy-urdine (dU) Uridine monophosphate(UMP)

A bit of nucleic acid nomenclature

* Can also have two or three phosphates, and de-oxy variety, too

B-DNA:

- right-handed

- most common form

- 0.34 nm rise

- 10.5 bp per turn

- 3.4 nm pitch

- adopted in aqueous

A-DNA:

- right-handed

- broader than B

- 0.26 nm rise

- ~11 bp per turn

- 2.8 nm pitch

- adopted in non-aqueous

- most common form for RNA

- has “hole” down the center

Z-DNA:

- left-handed

- zig-zaggy

- ~12 bp per turn

- adopted sometimes by (CG)n repeats.

cruciform

Triple-strand

The structure of DNA and RNA

RNA molecules are more variable and can adopt structures that resemble proteins (e.g. t-RNA below).

Aptamers are DNA and RNA molecules that fold into a 3D structure and bind substrates (much like proteins)

Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG) that we are working with (initially).

A quick aside: What are aptamers?

Aptamers (from apt: fitted, suited; Latin aptus: fastened)

• Oligonucleotides which have a demonstrated capability to specifically bind molecular targets with high affinity (KD

= 10-6 to 10-9 M).

• First described by Joyce1 (1989), Tuerk & Gold2 and Ellington & Szostak2 (1990).

• Binding properties depend on 3D structure and thus on sequence. 1 G. F. Joyce Gene 82: 83-87 (1989)

2C. Tuerk & L. Gold, Science 249, 505 (1990).

3A. D. Ellington & J. W. Szostak, Nature, vol. 346, pp. 818-822, 1990

Twist, rise and linking number in DNA

L, linking number: Number of times one edge of ribbon linked around other – topological property cannot change w/o cutting. (calculate by L = T + W)T, twist = winding of Watson around Crick – integrated angle of twist/2 along length, not an integer, necessarily (calculate by T = (number of base pairs/(base pairs/turn))W, writhe = wrapping of ribbon axis around itself – noninteger, geometric propertySupercoiling (Writhe) important in vivo (most DNA is slightly negatively supercoiled). = superhelical density

Note: There are topoisomerases to convert topoisomers. They can ‘remove a knot’ by breaking double-stranded DNA and re-ligating DNA. Mutated topoisomerases cause cancer.

L = T + W

= W/T

Sample problem

A circular, plectonemic (‘braided’) helix of DNA is in the B

form and has a total of 1155 basepairs.

1. What is the twist of the DNA?

2. The DNA has a superhelical density of -0.273. The DNA is

put into an alcohol solution and it takes the A form. What

is the W, T, L, and ?

Central dogma …continued

Translation (inside the ribosome (with help of tRNA):

Translation: making a peptide using mRNA as the coding template (peptide synthesis)

mRNA

mRNA … messenger RNA

tRNA … transfer RNA

Genetic Code (same in all organism)

UAU, UAC = Tyrosine

The structure of proteins1° structure: Amino acid sequence

– Twenty amino acids common to all organisms.

– Each has amino group, carboxyl group, R group and a hydrogen in tetrahedral symmetry. Almost all organisms have “L” chirality, but some virus have the mirror-image “D” chirality. (see board)

– Linked together by peptide bond. Peptide bond can be trans or cis.

– Proteins have prosthetic groups (e.g. heme) and amino acids can get modified (sugars, phosphates, etc).

– Two important angles: Φ: N-C bond, Ψ: C-C bond Ramachandran plot of allowed angles (dis-allowed due to steric hindrance).

The structure of proteins

– Given N amino acids, there are 20N different sequences. Sequence determines structure. If >20% homologous, probably similar structure. Converse not true: very different sequences can have similar structures.

– Hydrophobicity/hydrophilicity values [or “hydropathy” values, i.e. “strong feeling about”] determines protein folding. In aqueous environment, the core is hydrophobic, the surface is hydrophilic; in the membrane, both are hydrophobic.

– Kyte-Doolittle Scale – measure of hydrophobicity. Hydrophobicity is determined by measuring the energy Gtrans of transfering an amino acid from organic solvent (or vapor) to water (more in introduction-3).

• If Gtrans is positive – hydrophobic; if negative hydrophilic.

– There are charged and uncharged side chains. Proteins have net charge and pockets of positive and negative charges, salt bridges. Isoelectric point: pH where net charge of protein is 0.

1° structure: Amino acid sequence

aq

nonaq

ln , where P , mole fractiontransferG RT P


• 1° structure: A polymer with a unique amino acid sequence.

• There are twenty different amino acids

Charged amino acids

Positively charged

Negatively charged

Source: Kyte J & Dootlittle, RF; J. Mol. Biol. 157, 110 (1982)




Hydrophobic amino acids

Nonpolar (hydrophobic) amino acids, aromatic

Nonpolar (hydrophobic) amino acids, alkyl

Nonpolar (hydrophobic) amino acids





Uncharged, polar amino acids

Polar amino acids, aromatic

Polar amino acids, amines

Polar amino acids, disulfide with adjacent Cys

Polar amino acids



Alpha helix:

- right-handed helix

- 0.15 nm translation (rise)

- 100° rotation (twist)

- 3.6 residues/turn

- Pitch: 0.54 nm

- stabilized by H-bonds between NH and CO group (four residues up).

2° structure: alpha helix

-helix (© by Irvine Geis)

Biochemistry Voet & Voet

Red – oxygen

Black – carbon

Blue – nitrogen

Purple – R-group

White – C

Hydrogen-bonds between C-O of nth

and N-H group of n+4th residue.


Beta sheet:

- Can have parallel and anti-parallel

- Distance between residues: 0.35 nm

- H-bonds between NH and CO groups of adjacent strands stabilized structure.

2° structure: beta strand

Note: Color-in atoms for practice


Domains (are to 3° structure as sheets and helices are to +2° structure): Structurally or functionally defined, eg calmodulin, DNA binding domain

3° Structure: Overall 3-D structureNext time: pictures of peptide chains in fibrinogen moleculeUse sphere, ball and stick, ribbon representation

4° StructureNon-covalently linked 3° Structures (eg Hemoglobin )Homodimer vs hetero dimer, Hemoglobin is a heterotetramer

Higher Order Structure: Super secondary (+2°) structure: turns, -Hairpin, Greek Key, , , barrel

H-bonding disfavored in aqueous environment -sheets inside globular proteins (prionshelix sheet)

Example: Structure of Fibrinogen(look at this structure in Protein Data Bank)

Six polypeptide chains: 2 A(610 a.a.), 2 B(461 a.a.), and 2 (411 a.a.) (human numbering).

Trinodular: 2 external D nodules; central E nodule (N-termini)

Parts not resolved: loopy C- domain stretching back to E nodule (after residue 220), N-terminal of - and - chains (fibrinopeptides A and B) and N-terminal of -chain (2x96 residues), C-terminal of -chain(2x16 residues).

Dimensions: about 45 nm x 4.5 nm

17 disulfide bonds: within E nodule and braces at ends of the alpha helix coiled coils

E nodule D noduleD nodule

Crystal structure of Chicken Fibrinogen (2x 1364 a.a.) . Z. Yang, J. M. Kollman, L. Pandi, R. F. Doolittle, Biochemistry 40, 12515-12523 (2001)

b-hole

a-hole

Formation of Fibrin Fibers

+

thrombin

fibrinogen

fibrin

B

AFibrinogen

b

a

Fibrinopeptides A & B

Thrombin

Fibrin (protofibrils)

Protofibrilformation

Further lateral aggregation

Lateral aggregation and branching

SEM image (Hantgan) of fibrin clot (plus platelets)

10 m

AFM image (Guthold) of fibrin clot

10 m

Image: M. Kaga, P. Arnold; Voet & Voet, “Biochemisty”, Wiley & Sons, NewYork, 1990

The protein data bankAn Information Portal to Biological Macromolecular Structures

nearly 80,000 structures (Jan 2012)

Go to:


We’ll do some exercises related to the homework.


Regular class times: MWF 10-10:50 AM shapiro/biophysics12/ Instructors: (1) Professor Martin...

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