The long term objectives of this project are: 1) To understand the mechanism of how one protein can catalyze the folding of another; 2) To understand kinetic barriers to protein folding; 3) To understand relationships between amino acid sequence and protein structure and stability. Genetic, spectroscopic and calorimetric methods will be used to study the energetics of the unusual folding reaction of the serine protease subtilisin. The biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is auto-processed to create the mature form of the enzyme. Once processed, the native conformation of mature subtilisin is difficult to reach from the unfolded state. The folding reaction can be catalyzed in vitro, however, by the addition of the 77 amino acid proregion as a separate polypeptide. The mechanism of how the prodomain catalyzes subtilisin folding will be determined by studying the kinetic effects of mutations in subtilisin and the prodomain. Kinetic barriers to folding will be explored by studying the structure and stability of the trapped, unfolded forms of subtilisin. Finally the relationships between sequence and stability will be explored by using monovalent phage display of the prodomain in a procedure which selects for stabilizing mutations. Inefficient in vitro folding is a limiting factor in the production of many recombinant proteins of biomedical and biotechnological interest. Insight into the nature of the energetic barriers to folding and precise understanding of the mechanism of folding catalysis should lead eventually to the design of novel protein-specific foldases. Better knowledge concerning how to stabilize proteins should greatly advance the fields of protein engineering, protein structure prediction and de novo protein design.