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PROBING THE FREE ENERGY LANDSCAPE OF A FOLDING PROTEIN
BY MEANS OF ATOMIC FORCE MICROSCOPY STRETCHING
EXPERIMENTS Meeting EMBIO
project Wien, 21-24 May
2006
Florence group Dr. Francesca Sbrana Ph.D.Dr. Francesca Sbrana Ph.D.
CSDC-Department of Physics-University of Florence-Italy
Outline
Objectives
Experimetal set up: Atomic Force Microscopy
Single Molecule Stretching Experiment
Worm-Like Chain Model
The Sample: Titin protein
Results
Conclusions and Future Work
Objectives
Single Molecule Stretching Experiments by AFM
Extraction of information on protein folding with high throughput and efficiency
Optimisation of the Experimental Set -Up
The free energy landscape experienced by a real protein folding towards its native state
Investigation of the limit of applicability of Jarzynsky’s equality
Experimetal set up: Atomic Force Microscopy
Electronic control
Cantilever
Laser beam
Photodiode
PZT
Sample
UpDown
3D-Topographic Image
Force – Distance Curves
C2C12 cell
Single Molecule Stretching Experiment
JPK-NanoWizard® AFM
Stretching experiments on polymeric protin 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
Single Molecule Stretching Experiment
Resisitence at the extention the force rise
A domain begins to unfolds
The force increase until the protein unfold completely
The force drops
Zlatanova et al. Progress in Biophysics and Molecular Biology 74, (2000) 37-61
The Apparatus
The experiment were carried out in PBS at ambient temperature
Single Protein Folding Experiment with high
throughput and efficiencyInvestigation of the limit
of applicability of Jarzynsky’s equality
Strategic driving protocol of an
home built AFM, based on a
digital controller
To keep the tip-protein contact for a defined time
To perform multi stretching cycles on the same protein
Automatically move the tip over the sample if no protein attachment
Critical Points
High throughput and efficiency
Jarzynsky’s equality
“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”
WORM LIKE CHAIN model
Software for an automated
statistical analysis of the stretching
data
Continuous filament with resistance to bendingAverage length over which the directionbecomes random:persistence length LpTotal length of the unfolded polymer chain: Contour length LcEnd-to-end length x
Lp persistence length
Lc contour length
Z displacement
T temperaure
JPK-NanoWizard® AFM
Titin is a giant globular protein responsible for the passive elasticity of the cardiac muscles, and it is made by tandem repeat of several Ig – like modules.
We engineered this protein to obtain Ig-like domain chains with 4 and 8 monomers starting from module Ig27 (namely T4 and T8 fragments).
The Sample: Titin protein
M. S. Z. Kellermayer, H. L. Granzier, FEBS Lett., 380, 281-286 ( 1996)
Two cysteine residues at the C terminus
His6 tag inserted at the N terminus
Ig27 Ig32 Ig34
Protein adsorbed onto evaporated gold
surface
H. Lui et al. Biophysical Journal 79, (2000) 51-65
Ig27-Ig34
First Results
Ig27-Ig30
Lc=28nm
Lp=0.4 nm
Conclusion and Future Work
We plan to improve our AFM experimental set-up
To repeat single stretching experiment
on same protein and along a grid
Linear driving protocol towards
sinusoidal driving protocol
Commercial AFM protocol to stretch fragments of titin protein: T4 and T8
Critical points high throughput and efficiency Jarzynsky’s equality
Driving parameters chosen and modified opportunely
Thank you !!!
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