This proposal describes experimental studies of protein folding in vitro. Elucidating how the amino acid sequence determines structure; i.e., the protein folding problem, is a central issue in modern structural biology. Two recent developments have made this problem particularly timely. Massive sequencing efforts have led to an explosion in Dr. Raleigh's knowledge of the primary sequence of proteins. Unfortunately, it is still impossible to predict structure from sequence. In principle, a more thorough understanding of the folding process will aid efforts to decipher the code that links sequence and structure. A detailed understanding of the folding process will also aid efforts to rationally modify proteins to enhance desired properties. The growing realization that protein misfolding and aggregation play a role in a number of different diseases has also focussed attention on the folding problem. A lack of structural specificity or a breakdown in the cooperativity of folding can have severe physiological consequences, and partially folded states play an important role in pathophysiological protein aggregation. This proposal focuses on the experimental investigation of several key issues in protein folding: (1) the structural heterogeneity of partially folded states of proteins (2) the origins of cooperativity in folding (3) the role of subdomains in folding (4) the role of overall chain topology and specific interactions in rapid folding. The molten globule state is a key intermediate in folding. A protein dissection approach in combination with studies of mutants will be used to pinpoint key regions of the molten globule state of alpha-lactalbumin. It is now recognized that proteins can populate a wide range of different types of partially folded states. Comparative studies of different members of the alpha-lactalbumin/lysozyme family will provide information about the origins of the different types of partially folded states. Kinetic experiments will elucidate the steps involved in the cooperative formation of the native state from the molten globule. Kinetic experiments with three small, rapid folding, helical proteins will provide information about the fundamental steps in rapid folding.