Stability of the residual structure in unfolded BPTI in different conditions of temperature and solvent composition measured by disulphide kinetics and double mutant cycle analysis.

The folding funnel model proposes a clear description of the protein folding process. To test this model, additional data on the structures populated in different stages of folding and their influence on further folding are required. Here, we use the double mutant strategy and disulphide formation kinetics measurements to study the impact on folding of the residual structure in unfolded bovine pancreatic trypsin inhibitor (BPTI). We show how five amino acid residues stabilise a folding initiation site, possibly a beta-hairpin, and influence the shape of the upper region of the folding funnel in BPTI in different conditions of temperature and solvent composition. Our data provide experimental evidence for the mechanism by which a fast search for a proper chain topology is made possible early in the folding of proteins. The results apply to proteins in general, not necessarily just to disulphide bonded proteins, since cysteine residues are used here merely as reporter groups.