Transcript AFM force spectroscopy as a nanotool for early detection

1st Annual Unither Nanomedical and Telemedical
Technology Conference
Alexey V. Krasnoslobodtsev, PhD
AFM force spectroscopy as a nanotool
for early detection of misfolded protein.
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
Chemical
Stress
Misfolded
protein
Environmental
Stress
Chaperones
Native
folded
protein
Pathophysiological
Stress
Generic
Perturbations
Protein aggregation
Disease
Protein fibrils
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
Plaques and tangles
Parkinson’s
Lewy bodies
Huntington’s
intranuclear inclusions
Prion amyloid plaques
Amyotrophic lateral
sclerosis aggregates
Claudio Soto, 2003
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
Mechanism of aggregation
• Stress (environmental)
induced misfolding
generates “sticky”
aggregation prone
conformation
Normally
folded
protein
• Normally folded protein
interacts with misfolded
protein
Misfolded •
protein
Oligomers
Large aggregates and fibrils
Cycle multiplies copies of
misfolded (diseased)
proteins
• Goal - looking at the first
stage of aggregation
(dimerization) at a single
molecule level
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
Expression of the protein
Protein misfolding
Aggregation
Prevent aggregation of
misfolded proteins
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
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.
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.
AFM force spectroscopy
Contact of the tip
with sample surface
2
Approaching
1)
2)
Rupture event
Rupture force
3
4
5
3)
4)
5)
Tip retraction
Stretching the linkers
Bond rupture
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
•
A seven amino acid sequence within the N-terminal
domain is responsible for the aggregation of the whole
Sup35 protein
– Sequence: GNNQQNY
7GNNQQNY13
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.
Sup35 Aggregation at different pHs
(Environmental Stress)
Environmental
Stress
Misfolded 1
40
35
30
25
20
15
10
5
0
pH 5.6
8
Misfolded 2
pH 5.6
0
200
400
600
800
1000
6
pH 3.7
pH 3.7
Counts
4
2
0
16
14
12
10
8
6
4
2
0
Misfolded 3
0
400
600
800
1000
pH 2.0
0
pH 2
200
200
400
600
Force (pN)
Morphology of aggregation – different misfolding states that have different
strength of interactions?
800
1000
AFM force spectroscopy – nanotool for
detection of misfolded state.
700
Amyloid -β peptide
600
Force, pN
500
•
400
300
200
100
•
0
0
2
4
6
8
10
pH
•
•
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.
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
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).
O
N
O
Peptide
DNA
S
O
4
O
N
O
O
N
O
O
O
O
O
..Si O
N
N
4
O
O
DNA - SH
Peptide-SH
aqueous solution
in w ater
+
H
O
HO
O
O ..Si
N O
O
O
4
O
Si
HO
Si
2. Automated exchange of buffers containing drugs of interest.
3. Automated data analysis.
N
O
O ..Si
N O
Si
N
O
Beyond Force Spectroscopy
Dynamic Force Spectroscopy (DFS) measurements
DFS – measures kinetic parameters of dissociation reaction
1
2
k BT  r  x 
F
 ln
k k T 
x
 off B 
r – pulling velocity
(loading rate)
F1 < F2
Dynamic Force Spectroscopy
PP
koff
P+P
14
7
0
ΔG‡
xβ
Force
k BT  r  x 
F
 ln
k k T 
x
 off B 
Distance to
transition state
Dissociation
rate constant
Counts
Loading rate
Loading Rate
0
200
400
600
800
40
20
0
27
18
9
0
30
20
10
0
15
10
5
0
24
12
0
16
8
0
0
100
200
Force (pN)
ln r
300
1000
Dynamic Force Spectroscopy
GNNQQNY – Sup35 yeast prion
Rupture force, pN
350
300
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.
250
200
koff
150
100
50
0
1
10
2
10
10
3
10
4
Loading rate, pN/sec
10
5
Dynamic Force Spectroscopy
k1off
Rupture force, pN
350
k2off
0.2 Å
3.5 Å
300
250
200
koff
150
100
50
0
1
10
2
10
10
3
10
4
10
5
Loading rate, pN/sec
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.
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