1. Alexey V. Krasnoslobodtsev, PhD
1st Annual Unither Nanomedical and Telemedical Technology Conference
Alexey V. Krasnoslobodtsev, PhD
AFM force spectroscopy as a nanotool for early detection of misfolded protein.

2. Outline Misfolding (conformational) diseases – background.
Single molecule approach (Force spectroscopy) to study misfolding phenomenon.
Force spectroscopy - advantages and applications.
Beyond measuring forces of intermolecular interactions – Dynamic Force Spectroscopy.

3. Protein folding, misfolding and aggregation
Environmental
Stress
Misfolded
protein
Chemical
Stress
Chaperones
Native folded protein
Protein aggregation
Generic
Perturbations
Pathophysiological
Stress
Disease
Protein fibrils

4. Protein Misfolding (Conformational) Diseases
Many human diseases are now recognized to be conformational diseases associated with misfolding of the proteins and their consequent aggregation.
Alzheimer’s
Parkinson’s
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
Plaques and tangles
Lewy bodies
Huntington’s
intranuclear inclusions
Prion amyloid plaques
Amyotrophic lateral
sclerosis aggregates
Claudio Soto, 2003 4

5. 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
Misfolded
protein
Oligomers
Large aggregates and fibrils 5

6. Possible therapeutic interventions for protein misfolding diseases
Skovronovsky D.M., et al., 2006, Annu. Rev. Pathol. Dis., 1:151-70

7. Therapeutic approaches to misfolding diseases
Expression of the protein
Protein misfolding
Prevent aggregation of
misfolded proteins
Aggregation
Loss of neuronal function and cell death
Neurodegeneration
Small molecules that bind to specific regions of the misfolded protein and stabilize it.
Chemical (pharmacological) Chaperones

8. Rationale
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.
Rationale: A clear understanding of the molecular mechanisms of misfolding and aggregation will facilitate rational approaches to prevent protein misfolding mediated pathologies.

9. Probing interactions between individual molecules by AFM force spectroscopy
Dimerization of misfolded proteins is the very first step in aggregation process.
Force
AFM force spectroscopy allows studying:
Distance
Binding strengths - measures forces of interactions between individual molecules.

10. AFM force spectroscopy2)
Contact of the tip
with sample surface 1)
Approaching 2
Rupture event
Rupture force 3 4 5 3) 5) 4)
Tip retraction
Bond rupture
Stretching the linkers

11. Model system - 7 aa peptide from Sup35 yeast prion
Misfolding –
exposing “hot” regions
Aggregation
“Hot” regions are short stretches of peptide sequences.
Alzheimer’s: amyloid-beta peptide 1-40(42) -> Aβ16-22 is responsible for aggregation.
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 1
122
253
685
7GNNQQNY13
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,

12. Sup35 Aggregation at different pHs (Environmental Stress)
Misfolded 1
Misfolded 2
Misfolded 3
Environmental
Stress
pH 5.6
pH 3.7
pH 2
Morphology of aggregation – different misfolding states that have different
strength of interactions?

13. AFM force spectroscopy – nanotool for detection of misfolded state.
Amyloid -β peptide
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.

14. AFM force spectroscopy - High throughput screening machine for detecting efficient therapeutic agents
Control
Drug #1
Drug #2
Drug #3
Drug #2 is the best candidate for the development of effective therapeutic agents
Force of intermolecular interactions

15. Challenges 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).
Peptide
Peptide-SH
Automated exchange of buffers containing drugs of interest.
Automated data analysis.

16. Beyond Force Spectroscopy Dynamic Force Spectroscopy (DFS) measurements
DFS – measures kinetic parameters of dissociation reaction 1 2
r – pulling velocity
(loading rate)
F1 < F2

17. Dynamic Force Spectroscopyxβ
ΔG‡
PP P + P
koff
Loading Rate
Loading rate
Distance to
transition state
Dissociation
rate constant
Force
ln r 17

18. Dynamic Force Spectroscopy
GNNQQNY – Sup35 yeast prion
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. 18

19. Dynamic Force Spectroscopy
k1off
k2off
0.2 Å
3.5 Å
Two barriers in the energy profile:
Inner (second fit) and outer (first fit) activation barriers
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.
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.

20. Summary
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.
There is an intimate relationship between aggregation propensity (protein misfolding) and strength of interprotein interactions.
Force spectroscopy allows to study the mechanism of early dynamic events in the aggregation process which is not accessible by any other available method.
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.

21. 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