Early events in the disulfide-coupled folding of BPTI.
Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.
Bulaj
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Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.
Goldenberg
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DP
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D003080:Cold Temperature; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D013438:Sulfhydryl Compounds; D007611:Aprotinin; D003545:Cysteine