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.

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 The free energy landscape experienced by a real protein folding towards its native state Optimisation of the Experimental Set -Up Extraction of information on protein folding with high throughput and efficiency Investigation of the limit of applicability of Jarzynsky’s equality

Experimetal set up: Atomic Force Microscopy
PZT Laser beam
Electronic control

Photodiode Up Down Sample Cantilever

3D-Topographic Image

Force – Distance Curves

C2C12 cell

Single Molecule Stretching Experiment

JPK-NanoWizard® AFM

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

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

The Apparatus
Single Protein Folding Experiment with high throughput and efficiency Investigation of the limit of applicability of Jarzynsky’s equality

The experiment were carried out in PBS at ambient temperature

Strategic driving protocol of an home built AFM, based on a digital controller

Critical Points
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
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

Lp persistence length Lc contour length Z displacement T temperaure

Continuous filament with resistance to bending Average length over which the direction becomes random:persistence length Lp Total length of the unfolded polymer chain: Contour length Lc End-to-end length x
JPK-NanoWizard® AFM

Software for an automated statistical analysis of the stretching data

The Sample: Titin protein
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.
H. Lui et al. Biophysical Journal 79, (2000) 51-65

Ig27

Ig32

Ig34

His6 tag inserted at the N terminus

Two cysteine residues at the C terminus

Protein adsorbed onto evaporated gold surface

We engineered this protein to obtain Ig-like domain chains with 4 and 8 monomers starting from module Ig27 (namely T4 and T8 fragments).
M. S. Z. Kellermayer, H. L. Granzier, FEBS Lett., 380, 281-286 ( 1996)

First Results

Ig27-Ig30

Lc=28nm Lp=0.4 nm

Ig27-Ig34

Conclusion and Future Work
Commercial AFM protocol to stretch fragments of titin protein: T4 and T8

Critical points

high throughput and efficiency Jarzynsky’s equality

We plan to improve our AFM experimental set-up Driving parameters chosen and modified opportunely To repeat single stretching experiment on same protein and along a grid

Linear driving protocol towards
sinusoidal driving protocol

Thank you !!!

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