Post on 04-Feb-2016
description
AFM force spectroscopy as a nanotool for early detection of misfolded protein.
Alexey V. Krasnoslobodtsev, PhD
1st Annual Unither Nanomedical and Telemedical Technology Conference
Outline
1. Misfolding (conformational) diseases – background.
2. Single molecule approach (Force spectroscopy) to study misfolding phenomenon.
3. Force spectroscopy - advantages and applications.
4. Beyond measuring forces of intermolecular interactions – Dynamic Force Spectroscopy.
Protein folding, misfolding and aggregation
Protein aggregation
Chaperones
Protein fibrils
EnvironmentalStress
GenericPerturbations
ChemicalStress
Pathophysiological Stress
Disease
Native folded protein
Misfolded protein
Protein Misfolding (Conformational) Diseases
These diseases include neurodegenerative disorders such as Alzheimer’s, Parkinson’s disease, Huntington’s and prion diseases characterized by deposition of aggregates in Central Nervous System (CNS).
– Misfolded proteins are prone to aggregation
– Misfolded proteins and aggregates cause molecular stress and interfere with cellular function
Claudio Soto, 2003
Many human diseases are now recognized to be conformational diseases associated with misfolding of the proteins and their consequent aggregation.
Alzheimer’s
Plaques and tangles
Parkinson’s
Lewy bodies
Huntington’s intranuclear inclusions
Prion amyloid plaques
Amyotrophic lateral sclerosis aggregates
Mechanism of aggregation
• Stress (environmental) induced misfolding generates “sticky” aggregation prone conformation
• Normally folded protein interacts with misfolded protein
• Cycle multiplies copies of misfolded (diseased) proteins
• Goal - looking at the first stage of aggregation (dimerization) at a single molecule level
Normally folded protein
Misfoldedprotein
Large aggregates and fibrils
Oligomers
Possible therapeutic interventions for protein
misfolding diseases
Skovronovsky D.M., et al., 2006, Annu. Rev. Pathol. Dis., 1:151-70
Therapeutic approaches to misfolding diseases
Small molecules that bind to specific regions of the misfolded protein and stabilize it. Chemical (pharmacological) Chaperones
Expression of the protein
Protein misfolding
Aggregation
Loss of neuronal function and cell death
Neurodegeneration
Prevent aggregation of misfolded proteins
Rationale
Rationale: A clear understanding of the molecular mechanisms of misfolding and aggregation will facilitate rational approaches to prevent protein misfolding mediated pathologies.
Despite the crucial importance of protein misfolding and abnormal interactions, very little is currently known about the molecular mechanism underlying these processes.
Initial stages of misfolding and aggregation are very dynamic. High-resolution methods such as x-ray crystallography, NMR, electron microscopy, and AFM imaging have provided some information regarding the secondary structure of aggregated proteins and morphologies of self-assembled aggregates. But they are unable to characterize transient intermediates that can not be detected by these bulk methods.
We propose a novel method for identification and characterization of misfolded aggregation prone states of a protein as well as conditions favoring or disfavoring aggregation (misfolding). Single molecule force spectroscopy is capable of detecting interactions between transient species.
Probing interactions between individual molecules by AFM force spectroscopy
AFM force spectroscopy allows studying:
• Binding strengths - measures forces of interactions between individual molecules.
Force
Distance
Dimerization of misfolded proteins is the very first step in aggregation process.
AFM force spectroscopy
34
2
4)5)3)
1) Approaching 2)
Contact of the tip with sample surface
Tip retractionStretching the linkers Bond rupture
Rupture force
5
Rupture event
Model system- 7 aa peptide from Sup35 yeast prion
• A seven amino acid sequence within the N-terminal domain is responsible for the aggregation of the whole Sup35 protein
– Sequence: GNNQQNY
Nelson, R.R., Sawaya, M.R., Balbirnie, M., Madsen, A.Ø., Riekel, C., Grothe, R., Eisenberg, D. 2005. Structure of the cross-β spine of amyloid-like fibrils. Nature. Vol. 435, No. 9, 773-778.
1 122 253 685
7GNNQQNY13
Misfolding – exposing “hot” regions Aggregation
Alzheimer’s: amyloid-beta peptide 1-40(42) -> Aβ16-22 is responsible for aggregation.
“Hot” regions are short stretches of peptide sequences.
Huntington’s: polyQ (>40) -> elementary Q7 shows maximal kinetics of aggregation.
Parkinson’s: α-synuclein -> 12 aa regions is the core domain for aggregation.
Prion diseases: short peptide from Sup35 yeast prion
Sup35 Aggregation at different pHs (Environmental Stress)
pH 5.6
pH 3.7
pH 2
Misfolded 1
Misfolded 2
Misfolded 3
EnvironmentalStress
0 200 400 600 800 100002468
10121416 0 200 400 600 800 1000
0
2
4
6
8
pH 3.7
0 200 400 600 800 100005
10152025303540
pH 5.6
pH 2.0
Force (pN)
Co
un
tsMorphology of aggregation – different misfolding states that have different strength of interactions?
AFM force spectroscopy – nanotool for detection of misfolded state.
• Parallel circular dichroism (CD) measurements performed for Aβ peptide revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly β-sheet, structure at low pH.
• Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM.
• In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces.
• Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.
0 2 4 6 8 100
100
200
300
400
500
600
700
Fo
rce
, pN
pH
Amyloid -β peptide
AFM force spectroscopy -High throughput screening machine
for detecting efficient therapeutic agents
Drug #2 is the best candidate for the development of effective therapeutic agents
Force of intermolecular interactions
Drug #1
Drug #2
Drug #3
Control
Challenges
1. Robust system (for continuous measurements) We have recently developed surface chemistry which allows simple and reliable covalent attachment of biomolecules to the surfaces (AFM tip and mica).
2. Automated exchange of buffers containing drugs of interest.
3. Automated data analysis.
OH Si
O
O
ONOH
N OO
O
N OO
OOO
SiN
N OO4
O
N OO
4
SiO
O
ONOH
N OO
O
N OO
S
DNA
4
DNA - SH
+
in water
SiSi
..
..
aqueous solution
Si
..
Peptide-SH
Peptide
Beyond Force SpectroscopyDynamic Force Spectroscopy (DFS) measurements
Tkk
xr
x
TkF
Boff
B
ln1 2
F1 < F2
r – pulling velocity (loading rate)
DFS – measures kinetic parameters of dissociation reaction
Dynamic Force Spectroscopy
xβ
ΔG‡
PP P + Pkoff
Tkk
xr
x
TkF
Boff
B
ln
0 100 200 30008
16
Force (pN)
Cou
nts
01224
05
1015
0102030
09
1827
02040
0 200 400 600 800 100007
14
Load
ing
Rat
e
ln r
ForceDissociation rate constant
Distance to transition state
Loading rate
Dynamic Force Spectroscopy
101 102 103 104 1050
50
100
150
200
250
300
350
koff
Rup
ture
forc
e, p
N
Loading rate, pN/sec
A dynamic force spectrum at pH=2.0 reveals two linear regimes distinguishable by differences in slopes. This is usually attributed to a molecular dissociation of a complex that involves overcoming of more than one activation barrier.
GNNQQNY – Sup35 yeast prion
These data suggest that the ability of misfolded protein to form a stable dimer is a unique property of these conformational states for proteins suggesting a possible explanation for the phenomenon of the protein self-assembly into nanoaggregates.
Two barriers in the energy profile: Inner (second fit) and outer (first fit) activation barriers
Dynamic Force Spectroscopy
101 102 103 104 1050
50
100
150
200
250
300
350
koff
Rup
ture
forc
e, p
N
Loading rate, pN/sec
3.5 Å
0.2 Å
k2off
k1off
The estimated positions of inner and outer barriers are 0.2 and 3.5 Å. The off rates are 286 and 0.9 s-1.Estimated lifetime of a dimer is 1.1 s which is much longer than nano/microsecond conformational dynamics of a monomer.
Summary
1. Novel nanoprobing approach to study initial steps of misfolding and aggregation is proposed on the basis of AFM force spectroscopy operating on a single molecule level.
2. There is an intimate relationship between aggregation propensity (protein misfolding) and strength of interprotein interactions.
3. Force spectroscopy allows to study the mechanism of early dynamic events in the aggregation process which is not accessible by any other available method.
4. A dimer formed by two misfolded peptides is very stable as compared to monomer conformational dynamics providing the explanation for the phenomenon of the protein self-assembly into nanoaggregates.
Acknowledgements
• Yuri L. Lyubchenko, Ph.D., D. Sc.
• Lab Members:
– Luda Shlyakhtenko, Ph.D.
– Alex Portillo
– Jamie Gilmore
– Junping Yu, Ph.D.
– Mikhail Karymov, Ph.D.
– Shane Lippold
– Nina Filenko, Ph.D.
– Igor Nazarov, Ph.D.
– Alexander Lushnikov, Ph.D
Supported by NIH and Nebraska Research Initiative (NRI) grants to YLL