The molecular thermodynamics and kinetics of folding of the small globular protein T4 lysozyme will be determined by atomic force microscopy. Lysozyme will be covalently attached to the substrate and to the cantilever of an atomic force microscope; the distance between the cantilever and substrate will be increased, overcoming the weak forces which hold the protein in the folded state, causing the protein to unfold. The force vs. distance curve for unfolding the protein will be measured to study several properties of the unfolding, including the cooperativity of unfolding, the deformability of the folded state, the free energy and entropy of unfolding and the existence of unfolding intermediates. The deformability of the folded state will give information about the dynamics of the folded protein. The free energy of unfolding will be directly measured as the integral of force vs. distance. These properties will be determined for proteins attached through different residues, to determine the results of force denaturation from different sides of the protein. Molecular modelling of the results will be used to determine the pathway of unfolding.
