This proposal concerns two aspects of protein folding and stability: the nature of partially-folded species, especially the molten globule state, and the factors which determine a particular folding pathway. The long-term goals are to learn how the amino acid sequence of a polypeptide guides the folding to the native state, and determines the stability of protein conformations. A relatively compact protein conformational state, often referred to as the molten globule state, has been shown to be an intermediate in the refolding of certain proteins, and to be stable under certain denaturation conditions for some proteins. One of the goals of this research is to increase the understanding about the molecular details and physical properties of molten globules. The molten globule state will be studied under both equilibrium and transient refolding conditions. In addition the pathway of folding will be mapped out by using specific signals from different regions of the molecule in stopped-flow kinetics experiments at both ambient and subzero temperatures. A variety of biophysical techniques will be used to structurally characterize the molten globule state from several different proteins (in order to establish generalities); these include FTIR, circular dichroism (CD), fluorescence and hydrodynamic radius measurements. In addition kinetic experiments using stopped-flow fluorescence and CD will be performed.
