1. 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

2. 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

3. 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

4. Experimetal set up: Atomic Force Microscopy Electronic control Cantilever Laser beam Photodiode PZT Sample Up Down 3D-Topographic ImageForce – Distance Curves C2C12 cell

5. Single Molecule Stretching Experiment JPK-NanoWizard® AFM

6. 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

7. The Apparatus The experiment were carried out in PBS at ambient temperature Single Protein Folding Experiment with high throughput and efficiency Investigation of the limit of applicability of Jarzynsky’s equality Strategic driving protocol of an home built AFM, based on a digital controller

8. 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”

9. WORM LIKE CHAIN model Software for an automated statistical analysis of the stretching data 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 Lp persistence length Lc contour length Z displacement T temperaure JPK-NanoWizard® AFM

10. 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 Ig27Ig32Ig34 Protein adsorbed onto evaporated gold surface H. Lui et al. Biophysical Journal 79, (2000) 51-65

11. Ig27-Ig34 First Results Ig27-Ig30 Lc=28nm Lp=0.4 nm

12. 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 pointshigh throughput and efficiency Jarzynsky’s equality Driving parameters chosen and modified opportunely

13. Thank you !!!