Reconstructing protein folding freeenergy landscape from AFM stretching experiments

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  Reconstructing protein folding free-energy
landscape from AFM stretching experiments
Biophys07 Arcidosso, 03-05 September 2007
Dr. Francesca Sbrana Ph.D.
ISC-CNR, CSDC-Department of Physics-University of
Florence-Italy
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Summary
Single Molecule Stretching Experiments by AFM
To force mechanically a protein to unfold the
rapture force -kinetic information reconstructi
on of the free energy landscape
Application of Jarzynskis Equation
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The Sample Titin protein
Titin is a giant globular protein responsible for
the elasticity of the cardiac muscle
H. Lui et al. Biophysical Journal 79, (2000)
51-65
Protein adsorbed onto evaporated gold surface
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Atomic Force Microscopy
3D- Topography image
Force-Distance Curve
C2C12 cell
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Single Molecule Stretching Experiment

• Resistance at the extension the force rises
• A domain begins to unfolds
• The force increase until the protein unfold
  completely
• The force drops

Stretching experiments on polymeric protein
result in force-distance curves showing a
characteristic sawtooth pattern The peaks of the
sawtooth pattern correspond to the consecutive
mechanical unfolding of individual domains
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Extraction of structural information
Lp persistence length Lc contour length Z
displacement T temperaure
WORM LIKE CHAIN model
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Free Energy Landscape from Jarzynski Eq.
The free energy landscape between two
equilibrium states is well related to the
irreversible work required to drive the system
from one state to the other
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Free Energy Landscape from Jarzynski Eq.
Extended JE in terms of molecular elongation ( l )
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Extraction of kinetic information
k 0.04 N/m
xu 0.21 0.04 nm
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Free Energy Landscape from Jarzynski Eq.
Free energy landscape F as a function of the
molecular elongation l for different values probe
velocity, v 200, 400, 1000, 2000 nm/s
Inset Plot of F in the small l regime
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Free Energy Landscape from Jarzynski Eq.
Free energy landscape F(l, f ) for three values
of the force with v 200 nm/s.
Stepwise unfolding events for Ig2712 using force
clamp method. The step increases in length are
plotted as a function of the applied force
A.F. Oberhauser et al. PNAS, 98 (2001), 468-472
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The Apparatus
EMBIO-AFM
Strategic driving protocol of an home built AFM,
based on a digital controller
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Examples of Force-Distance Curves
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Software Optimization

• To perform maps of force-distance curve
• (ex. 8 x 8 pixel64 curves)
• To keep the tip-protein contact for a defined time

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Software Optimization
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Software Optimization
Intelligent algorithm
approach to surface, v1
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stay in contact for t1
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withdraw out contact, v2
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reach a prestabilized z1 position out contact
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withdraw again and stop
has deflection a certain value?
yes
approach again
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Hardware Optimization
Low Coherence laser diode
to reduce uncertainty in the measurements of the
force due to beam interference
Fast and Linear scanner
to increase accuracy in the nano positioning of
the probe respect to the sample
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Conclusion

• Application of Jarzynskis Equation

Single Molecule Stretching Experiments by AFM
Reconstruction of the free energy landscape
To project force clamp spectroscopy experiment
Direct Kinetic information
We plan to perform measurements with our
optimised AFM experimental set-up
To study other proteins Elastin, Fibrinogen
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Collaborations
Jarzynski Equality Alberto Imparato, Luca Peliti
Univ. Federico II Napoli Control design Paolo
Baschieri, Cesare Ascoli IPCF CNR Pisa
Michele Basso, Roberto Genesio, Donatello
Materassi Syst. Inform. Dip. Univ. Firenze,
Protein folding modeling Lorenzo Bongini, Lapo
Casetti, Carlo Guardiani, Roberto Livi, Stefano
Luccioli, Alessandro Torcini Dip. Fisica, Univ.
Firenze, Center for Complex Dynamics,
ISC-CNR-Firenze Nanoscopy Group-
ISC-CNR-FIRENZE Franco Quercioli, Francesca
Sbrana, Bruno Tiribilli, Massimo Vassalli,
Raffaella Mercatelli
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Thank you !!!