Extensive Nonrandom Structure in Reduced and Unfolded Bovine Pancreatic Trypsin Inhibitor

Hong Pan, Elisar Barbar, George Barany, Clare Woodward

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Abstract

Two-dimensional 1H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 °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 α-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol. The indications of numerous turn-like conformations and of hydrophobic interactions are consistent with earlier reports that reduced BPTI is a molten coil which is collapsed to some extent but not as much as native, and which has exposed, clustered hydrophobes [Ferrer, M., Barany, G., & Woodward, C. (1995) Nature Struct. 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-[13C]methylcysteine, also displays chemical shift dispersion in HMQC-detected resonances of the [13C]methyl protons. The dispersion is lost by addition of guanidine hydrochloride, indicating that nonrandom structure in reduced BPTI is disrupted by the denaturant.

Original languageEnglish (US)
Pages (from-to)13974-13981
Number of pages8
JournalBiochemistry
Volume34
Issue number43
DOIs
StatePublished - Oct 1995

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