There are a large number of proteins whose three dimensional structures have been determined. However, virtually very little is known as to how these proteins are folded. In order to elucidate the molecular mechanisms of protein folding, the role of the pro-sequence in the folding of subtilisin E, an alkaline serine protease produced by Bacillus subtilis has been studied. This protein is produced from the pre-pro subtilisin consisting of the pro-sequence (signal peptide) of 21 residues, the pro-sequence of 77 residues, and the mature protease of 275 residues. The pro-sequence is absolutely essential for the production of enzymatically active subtilisin, and functions as an intramolecular chaperon for protein folding. This is a novel, unprecedented finding and provides and ideal and unique system for studying the molecular mechanisms of protein folding. In this application the exact role of the pro-sequence in the folding pathway will be elucidated. It is of interest to investigate how the pro-sequence interacts with mature, active subtilisin and pursue biochemical and biophysical characterization of pro-subtilisin and of pro- peptides of various sequences and lengths. By using molecular genetic approaches as well as synthetic pro-peptides, the site or regions responsible for refolding denatured subtilisins will be determined. The goal in this proposal is to elucidate the precise roles of the pro-peptide in the folding of subtilisin, which is expected to provide important insight into understanding of the molecular mechanisms of protein folding. For this purpose, the following experiments will be performed: 1. Isolation of mutant pro-subtilisins defective in autoprocessing and their characterization; 2. Isolation and characterization of mutations within the pro-peptide defective in producing active subtilisin; 3. Isolation of second site suppressor mutations a new method developed in the laboratory will be extensively employed using localized PCR random mutagenesis; 4. Characterization of folding intermediated accumulated as a result of mutations; 5. Production of the wild-type and mutant pro-peptides by organic synthesis and by recombinant DNA technology. Their biochemical an biophysical characterization and their interaction with mature subtilisin.
