Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor.

Abstract

Two-dimensional 1H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 degrees C, have been assigned. Spectra indicate considerable conformational averaging, as expected for a flexible, unfolded protein. The presence of extensive nonrandom structure is detected by the presence of NHi-NHi + 1 and aromatic-aliphatic NOEs. Sequential amide-amide NOEs indicate that turn-like conformations are significantly populated at 18 pairs of residues along the chain. Many of these are located in a turn, loop, or helix in native BPTI, but six are observed for contiguous pairs in the segment composed of residues 29-35, which in native BPTI constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic interactions. Multiple aromatic-aliphatic NOEs are observed for pairs of residues that are within 1-3 residues of each other. Most are non-native and involve residues in both strands of the central antiparallel strand-turn-strand of native BPTI comprised of residues 18-35. All NOEs reported for oligopeptides spanning the BPTI sequence [Kemmink, J., & Creighton, T. (1993) J. Mol. Biol. 234, 861-878] are observed in reduced BPTI, but many others are present as well. Similar spectra are obtained for naturally occurring BPTI reduced by dithiothreitol, BPTI with cysteines replaced by alpha-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)