Sequence-dependent folding of single proteins under mechanical load

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Proteins must fold into precise three-dimensional structures in order to function correctly. The folding process is an essential part of the flow of genetic information in the cell from storage to useful deployment. Despite many decades of research on protein folding, much remains to be learned about how the amino acid sequence affects folding, due to the experimental challenges in measuring the folding transition directly. This project will probe the basic principles of folding by using mechanical force applied by an optical trap to unfold and refold single protein molecules repeatedly. Individual folding trajectories will be measured with high-precision by monitoring the molecular end-to-end distance under both equilibrium (constant force) and non-equilibrium (changing force) conditions. Quantitative thermodynamic and kinetic properties of the folding process will be determined from these trajectories, including the free energy for folding, the folding and unfolding rates as a function of force, the presence and location along the reaction coordinate of intermediate states, and the location and height of energy barriers. The effect of sequence on these properties will be investigated systematically by varying the amino acid sequence of simple protein domains.