title: Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor.
abstract: 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](Abu). Two new analogues with modifications in the beta-turn, P26D27[14-38](Abu) and N26G27K28[14-38](Abu), are compared to partially folded [14-38](Abu), as well as to [R](Abu), the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of [14-38](Abu) 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](Abu), but while P26D27[14-38](Abu) has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K28[14-38](Abu). Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both [14-38](Abu) and P26D27[14-38](Abu) significantly inhibit modified trypsin. N26G27K28[14-38](Abu) has low but measurable inhibitor activity, while [R](Abu) has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14-38](Abu) and by both variants but not by [R](Abu). 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](Abu) and both variants, as well as [R](Abu), 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](Abu) and P26D27[14-38](Abu) and is probably native-like for N26G27K28[14-38](Abu). [R](Abu) also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.
authors.affiliation: Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, Minnesota 55455, USA.
authors.forename: Judit
authors.initials: J
authors.lastname: Tulla-Puche
authors.forename: Irina V
authors.initials: IV
authors.lastname: Getun
authors.forename: Clare
authors.initials: C
authors.lastname: Woodward
authors.forename: George
authors.initials: G
authors.lastname: Barany
mesh_terms: D019943:Amino Acid Substitution; D000613:Aminobutyrates / Q000737:chemistry*; D000818:Animals; D007611:Aprotinin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004795:Enzyme Stability; D008744:Methylamines / Q000737:chemistry; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry
chemical_list: D000613:Aminobutyrates; D008744:Methylamines; D020033:Protein Isoforms; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; C033859:2-aminobutyric acid methyl ester; D007611:Aprotinin; C005855:trimethyloxamine; D003545:Cysteine