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 !!!