Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor.

Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by alpha-amino-n-butyric acid (Abu) while retaining the disulfide between Cys 14 and 38; the resultant variant is termed 14-38. Two new analogues with modifications in the beta-turn, P26D2714-38 and N26G27K2814-38, are compared to partially folded 14-38, as well as to R, the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of 14-38 have been determined by circular dichroism (CD), one-dimensional (1)H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Both analogues are more disordered than the parent 14-38, but while P26D2714-38 has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K2814-38. Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both 14-38 and P26D2714-38 significantly inhibit modified trypsin. N26G27K2814-38 has low but measurable inhibitor activity, while R has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by 14-38 and by both variants but not by R. We conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable structure, contain minor populations of native-like conformations. Partially folded 14-38 and both variants, as well as R, have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamine N-oxide (TMAO). TMAO-induced structure is formed cooperatively, as indicated by thermal unfolding curves. Inhibitor activity as a function of TMAO concentration implies that the osmolyte-induced structure is native-like for 14-38 and P26D2714-38 and is probably native-like for N26G27K2814-38. R also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.