The folding of proteins in the cell is determined by complex interactions with cellular components and proceeds with rates and efficiency much greater than those observed in vitro. Folding is a key step in protein biogenesis; mutations that affect protein folding lead to cellular abnormalities that, in humans, are linked to several disease states. Because of the considerable technical problems that complicate in vivo studies, little is known about the physiological role of the enzymes that catalyze rate limiting conformational steps in proteins, or on how the protein biosynthetic machinery affects the folding pathway. In earlier work, we demonstrated that the study of the folding kinetics of Bovine Pancreatic Trypsin Inhibitor (BPTI) in the periplasmic space of E.coli represents an excellent model for delineating the role of cellular factors on protein folding. Specific advantages of this experimental system include: (i) the detailed information, available from in vitro studies, on the stability and conformation of BPTI folding intermediates; (ii) the wealth of genetic techniques for E.coli; (iii) the ease with which disulfide bond formation and isomerization in BPTI can be monitored in vivo; (iv) the fact that the oxidative folding of BPTI in the periplasmic space involves all known components of the periplasmic oxidoreductase machinery. In this work, we now propose to examine the following issues: 1. The detailed role of all the components of the Dsb system (DsbA, DsbB, DsbC, DsbD and DsbE, the proteins that mediate disulfide bond formation in bacteria) on: (i) the oxidation, disulfide isomerization and formation of native BPTI in vivo, and on: (ii) the redox state of the periplasmic space, will be determined. The effect of the external redox environment and of the growth pH on the folding pathway will be analyzed. In addition, conditions that enhance the rate of disulfide bond isomerization in the periplasmic space will be sought. 2. The relation between export across the cytoplasmic membrane and folding will be investigated in detail. Specific issues that will be addressed include how the leader peptide affects the folding pathway and whether protein oxidation commences in the cytoplasm. 3. The role of the pro region, a 13 amino acid N-terminal extension that is encoded by the BPTI cDNA and has been shown to affect the folding pathway of the mature protein in vitro, will be evaluated. Finally, the role of amino acid substitutions that have been shown to exert a pronounced effect on the in vitro folding of BPTI kinetics will now be investigated in the cellular context.
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