STABILITY AND FOLDING OF BPTI WITH UNNATURAL CROSSLINKS

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Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue small protein with three disulfide bridges. BPTI has been used extensively in protein folding studies, which suggest that the folding pathway proceeds via intermediates containing specific disulfide bonds. Formation of any one disulfide will stabilize the slow-exchange core and initiate folding by destabilizing the fully extended unfolded form. Our hypothesis, which we seek to test by total synthesis, is that any natural or unnatural cross- link that does not introduce unfavorable strain, will have the same effect. One type of cross-link is to circularize BPTI, which may be possible because in the solution structure of BPTI, the N- and C-termini are in close three-dimensional proximity. The specific aims of this proposal are to synthesize and characterize circularly permuted variants of BPTI and analogues with unnatural cross-links. This work will provide definitive conclusions about the roles of disulfide bridges in BPTI folding and stability and is expected to lead to significant insights that contribute to our understanding of the critical factors governing proper folding of proteins.