Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor.
We have shown previously that during the oxidative folding of bovine pancreatic trypsin inhibitor only intermediates with native disulfide bonds are well populated. Nevertheless, these studies also confirmed the earlier conclusion [Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293] that the rate-limiting transition in the kinetically preferred route for folding involves intramolecular disulfide bond rearrangements. Consequently, intermediates with nonnative disulfide bonds must form transiently during folding. Two specific nonnative species, denoted [30-51; 5-14] and [30-51; 5-38], in which numbers indicate residues participating in a disulfide bond, can be detected at low levels in kinetic folding experiments with bovine pancreatic trypsin inhibitor. By working with purified reversibly trapped intermediates, the role of these two nonnative species has been examined directly. These species are found to be in relatively rapid exchange with each other and with an initially formed native two-disulfide intermediate [30-51; 14-38]. Thus, the low abundance of the two nonnative species detected in kinetic folding experiments reflects primarily their low thermodynamic stability as compared to this native intermediate. To a small extent, these nonnative species form the productive native intermediate [30-51; 5-55], which is the immediate precursor to the native protein. However, an equal amount of [5-55; 14-38], a nonproductive dead-end intermediate, is also produced. Thus, the nonnative species detected during the folding of bovine pancreatic trypsin inhibitor are not committed to forming the productive native intermediate, nor do they serve to direct folding specifically toward a productive route.
Weissman
J S
JS
Howard Hughes Medical Institute, Department of Physics, Nine Cambridge Center, MA 02142.
Kim
P S
PS
D000818:Animals; D007611:Aprotinin / Q000648:ultrastructure*; D002417:Cattle; D003461:Crystallography; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D013438:Sulfhydryl Compounds; D007611:Aprotinin