DYNAMICS OF THE CONFORMATIONAL ENSEMBLE OF PARTIALLY FOLDED BOVINE PANCREATIC TRYPSIN-INHIBITOR

A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPT I), [14-38](Abu), is a partially folded ensemble which includes two, a nd in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and h eteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoe s local motions that are slow, noncooperative, independent fluctuation s of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to meas ure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly sm aller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. Th e reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antip arallel beta-sheet. Global unfolding interconversion rate constants va ry over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T-1, T-2, and N OE relaxation parameters, are obtained for the slowly interchanging co nformations in the partially folded ensemble. Clear differences are ob served between the two conformations; one is more flexible and less co mpact than the other. In the more flexible and disordered partially fo lded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These s ame sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formati on of a partially folded ensemble in which the slow-exchange core is t he most stable region and in which segmental fluctuations reflect mult iple nuclei for folding of the rest of the molecule.