Dynamic structure of a highly ordered beta-sheet molten globule: multiple conformations with a stable core.

The structure of [14-38]_Abu, a variant of bovine pancreatic trypsin inhibitor (BPTI) with only the 14-38 disulfide bridge intact, has been analyzed by two-dimensional ¹H and ¹H- ¹⁵N NMR. Except for the 18-24, 29-35 antiparallel β-sheet, residues in all regions of the molecule give two exchange cross peaks for each ¹H; for one residue, Gly 37, three exchange cross peaks are observed. The presence of exchange cross peaks indicates that the residues sample conformations that interconvert on a time scale of milliseconds or longer. Over 90% of the NMR spectra have been assigned, including backbone and side chain atoms and their exchange cross peaks. Analyses of chemical shifts, chemical exchange, hydrogen isotope exchange, and NOEs indicate that [14-38]_Abu at pH 4.5 and 1°C is an ensemble of interconverting conformations, in all of which the 18-24, 29-35 antiparallel β-sheet is native-like and intact. Outside the antiparallel β-sheet, residues undergo local order/disorder transitions. The stable structure of [14-38]_Abu is not in the vicinity of the 14-38 disulfide bond but rather is in the slow-exchange core. NOE analysis indicates that the main tertiary interactions involve hydrophobic contacts with the rings of Tyr 21, Tyr 23, and Tyr 35. As a model for early folding intermediates, the structure of [14-38]_Abu suggests that BPTI folding is initiated by stabilization of a turn existing in the unfolded protein and involves both local and nonlocal hydrophobic interactions.