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 °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.