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](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.
Tulla-Puche
Judit
J
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, Minnesota 55455, USA.
Getun
Irina V
IV
Woodward
Clare
C
Barany
George
G
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
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