Native-like Conformations Are Sampled by Partially Folded and Disordered Variants of Bovine Pancreatic Trypsin Inhibitor
详细信息 查看全文
- 作者：Judit Tulla-Puche ; Irina V. Getun ; Clare Woodward ; and George Barany
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：February 17, 2004
- 年：2004
- 卷：43
- 期：6
- 页码：1591 - 1598
- 全文大小：139K
- 年卷期：v.43,no.6(February 17, 2004)
- ISSN：1520-4995
文摘
Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) areaccessed by replacing Cys 5, 30, 51, and 55 by 
-amino-n-butyric acid (Abu) while retaining the disulfidebetween Cys 14 and 38; the resultant variant is termed [14-38]Abu. Two new analogues with modificationsin the 
-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. Structuralfeatures of the new analogues of [14-38]Abu have been determined by circular dichroism (CD), one-dimensional 1H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Bothanalogues are more disordered than the parent [14-38]Abu, but while P26D27[14-38]Abu has a smallpopulation 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, thatminimizes cleavage of unfolded peptides. Both [14-38]Abu and P26D27[14-38]Abu significantly inhibitmodified trypsin. N26G27K28[14-38]Abu has low but measurable inhibitor activity, while [R]Abu has noactivity 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-likeconformations. Partially folded [14-38]Abu and both variants, as well as [R]Abu, have enhanced negativeellipticity 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 asa 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 alsoshows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsedand non-native.
-amino-n-butyric acid (Abu) while retaining the disulfidebetween Cys 14 and 38; the resultant variant is termed [14-38]Abu. Two new analogues with modificationsin the -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. Structuralfeatures of the new analogues of [14-38]Abu have been determined by circular dichroism (CD), one-dimensional 1H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Bothanalogues are more disordered than the parent [14-38]Abu, but while P26D27[14-38]Abu has a smallpopulation 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, thatminimizes cleavage of unfolded peptides. Both [14-38]Abu and P26D27[14-38]Abu significantly inhibitmodified trypsin. N26G27K28[14-38]Abu has low but measurable inhibitor activity, while [R]Abu has noactivity 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-likeconformations. Partially folded [14-38]Abu and both variants, as well as [R]Abu, have enhanced negativeellipticity 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 asa 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 alsoshows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsedand non-native.