The long range objective of the proposed work is to characterize the forces which stabilize the different conformational states of proteins. These states are thought to be determined mainly by a balance between hydrophobic forces which drive the protein toward condensed compact conformations and conformational entropy which favors extended, flexible conformations. This project focuses on aspects of the hydrophobic interactions which remain poorly understood. When a protein unfolds, nonpolar amino acids, originally segregated in the protein interior, are exposed to water. The thermodynamic consequences of this exposure can be modeled by transfer of small molecules from nonpolar environments to water. These studies and also theoretical analyses of water structure well account for the striking heat capacity changes which are observed when a protein unfolds. Similar models do not, however, account for the volume and compressibility changes observed under analogous conditions. The latter represents a substantial defect in understanding the thermodynamics of protein folding and is the main subject of the proposed work. The rationale of the approach is based on the supposition that different kinds of structural changes occur during the different phases of folding kinetics. Using an instrument developed in the laboratory the overall volume change is to be decomposed into components associated with the different kinetic phases of folding. This will allow partial volume changes associated with different kinds of structural changes to be characterized separately. Measurements of partial volume change will be supplemented with measurements of changes of heat capacity, compressibility and of the equilibrium folding transition. Additional major aims are to characterize the change in protein area exposed to interaction with water during unfolding and to explore further the applicability of solvent transfer measurements as models of protein volume and compressibility changes. Preliminary measurements on cytochrome c have demonstrated the feasibility of many of the proposed studies and will be extended in the proposed work.
