Early events in the disulfide-coupled folding of BPTI

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibi tor (BPTI) have shown that a previously unidentified intermediate with a si ngle disulfide is formed much more rapidly than any other one-disulfide spe cies. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable tha n the other two intermediates with single native disulfides. To characteriz e the role of the [14-38] intermediate and the factors that favor its forma tion, detailed kinetic and mutational analyses of the early disulfide-forma tion 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 loc al electrostatic effects, which enhance the reactivities of the Cys14 and 3 8 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 t he 14-38 disulfide, but the majority of these molecules then undergo intram olecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replaceme nts, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynam ic stability, even though many of the same substitutions greatly destabiliz ed the native protein and other disulfide-bonded intermediates. In addition , those replacements that did decrease the steady-state concentration of [1 4-38] did not adversely affect further folding and disulfide formation. The se 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.