Recent discoveries have shown that the pathway of protein folding in vivo is modulated by cellular processes such as interactions with chaperonins and enzymes which catalyze rate limiting conformational changes (foldases). These processes play a central role in protein assembly and transport, in morphogenesis and in cellular adaptation. Despite its importance, the mechanism of protein folding in vivo has not yet been subjected to detailed biochemical analysis. For this reason there is very little information on the detailed kinetics of folding in physiological environments or the in vivo role of cellular components that influence the native conformation. The technical problems that have hindered progress in this area can be circumvented by examining the folding of secreted proteins in the periplasmic space of E. coli. The Bovine Pancreatic Trypsin Inhibitor (BPTI) a protein whose folding in vitro has been characterized in great detail will be used as a model system. Because of the permeability of the outer membrane, the folding of BPTI secreted in the periplasmic space can be quenched chemically without disrupting the cell. Folding intermediates will be trapped by two complementary techniques: 1) Direct carboxymethylation of secreted BPTI by reacting with a high concentration 0.5 M iodoacetate. 2) Rapid acid-quenching to prevent disulfide bond formation followed by urea, neutralization and carboxymethylation in order to block free thiols irreversibly. To monitor the kinetics of folding, cells will be pulse labelled with radioactive amino acids and folding intermediates will be trapped at different times as described above. Native and partially folded BPTI species will be separated by immunoprecipitation and quantified by non-denaturing electrophoresis. the position of any disulfide bonds that have formed prior to quenching will be determined using a modification of well established procedures which have been employed by Creighton and coworkers for in vitro studies. The folding pathway of BPTI will be determined in mutants deficient in periplasmic foldases such as DsbA, the E. coli protein disulfide isomerase, and the periplasmic enzyme that exhibits proline isomerase activity. The latter study will be conducted using Cys30->Ala, Cys51- >Ala BPTI which has been reported to exhibit a rate limiting proline isomerization step in vitro. Comparison of the kinetics of accumulation of folding intermediates in mutant and wild type cells will help identify the rate limiting steps in vivo and provide an estimate of the rate acceleration afforded by foldases in the cell. Experiments will also be conducted to elucidate the role of transport across the cytoplasmic membrane on the folding of the mature protein in the E. coli periplasmic space. This work will involve determination of the folding kinetics of BPTI exported via defective leader peptides and the use of strains carrying mutations in late acting components of the secretory apparatus (particularly prlA alleles). Finally, the effect of physiological pH and redox potential in the periplasmic space will be analyzed.
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