EXTENSIVE NONRANDOM STRUCTURE IN REDUCED AND UNFOLDED BOVINE PANCREATIC TRYPSIN-INHIBITOR

Two-dimensional H-1 NMR spectra of an analog of reduced BPTI at pH 4.5 , 1 degrees C, have been assigned. Spectra indicate considerable confo rmational 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 Is pairs of residues along the chain. Many of these are located in a t urn, loop, or helix in native BPTI, but six are observed for contiguou s pairs in the segment composed of residues 29-35, which in native BPT I constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic in teractions. Multiple aromatic-aliphatic NOEs are observed for pairs of residues that are within 1-3 residues of each other. Most are non-nat ive and involve residues in both strands of the central antiparallel s trand-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 redu ced BPTI, but many others are present as well. Similar spectra are obt ained for naturally occurring BPTI reduced by dithiothreitol, BPTI wit h cysteines replaced by alpha-amino-n-butyric acid, and BPTI mutant F4 5A reduced by dithiothreitol. The indications of numerous turn-like co nformations and of hydrophobic interactions are consistent with earlie r reports that reduced BPTI is a molten coil which is collapsed to som e extent but not as much as native, and which has exposed, clustered h ydrophobes [Ferrer, M., Barany, G., & Woodward, C. (1995) Nature Struc t. Biol. 2, 211-217]. Comparison of the NOEs in reduced BPTI to those in a model for early BPTI folding intermediates suggests a significant role for non-native interactions in initial steps: of BPTI folding. A variant of reduced BPTI, in which all cysteines are replaced with S-[ C-13]methylcysteine, also displays chemical shift dispersion in HMQC-d etected resonances of the [C-13]methyl protons. The dispersion is lost by addition of guanidine hydrochloride, indicating that nonrandom str ucture in reduced BPTI is disrupted by the denaturant.