Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor.
Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor.
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天然样构象是通过牛胰蛋白酶抑制剂的部分折叠和无序变体进行采样的。
DOI：
10.1021/bi035301a
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Barany,George
中科院分区：
文献类型：
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作者：
Tulla-Puche,Judit;Getun,IrinaV;Woodward,Clare;Barany,George
通过α-氨基正丁酸（Abu）取代Cys 5、30、51和55，同时保留Cys 14和38之间的二硫基团，获得了部分折叠的牛胰蛋白酶抑制剂（BPTI）构象群；由此产生的变体被称为[14−38]Abu。在β-旋上进行修饰的两个新的类似物P26D27[14−38]Abu和N26G27K28[14−38]Abu与部分折叠的[14−38]Abu和未折叠的6个Cys残基全部被Abu取代的[R]Abu进行了比较。通过圆二色性（CD）、一维1h NMR和8-苯胺-1-萘磺酸（ANS）荧光实验确定了新的[14−38]abu类似物的结构特征。这两种类似物都比亲本[14−38]Abu更无序，但P26D27[14−38]Abu有少量的原生构象，而N26G27K28[14−38]Abu没有检测到有序结构。胰蛋白酶抑制试验使用改良的大鼠胰蛋白酶C191A/C220A进行，该酶能最大限度地减少未折叠肽的裂解。[14−38]Abuand P26D27[14−38]均显著抑制修饰胰蛋白酶。N26G27K28[14−38]abuh具有较低但可测量的抑制活性，而[R] abuh即使在相对于胰蛋白酶非常高的摩尔过量时也没有活性。[14−38]Abu和[R]Abu对ANS荧光均有增强作用。我们得出的结论是，部分折叠的BPTI，即使是那些很少或没有CD或核磁共振可检测结构的BPTI，也含有少量的天然类构象。部分折叠的[14−38]Abu和[R]Abu在渗透电解质三甲胺氧化物（TMAO）存在下的CD光谱中具有增强的负椭圆性。热展开曲线表明，tmao诱导结构是协同形成的。抑制剂活性作为TMAO浓度的函数表明，渗透诱导的结构类似于[14−38]Abuand和P26D27[14−38]Abuand可能类似于N26G27K28[14−38]Abu。[R] abuu也显示在TMAO存在下cd检测到的结构增加，但这种结构可能是坍塌的和非原生的。
Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by α-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 β-turn, P26D27[14−38]Abuand 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]Abuhave been determined by circular dichroism (CD), one-dimensional1H 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]Abuhas 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]Abuand P26D27[14−38]Abusignificantly inhibit modified trypsin. N26G27K28[14−38]Abuhas low but measurable inhibitor activity, while [R]Abuhas no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14−38]Abuand 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]Abuand both variants, as well as [R]Abu, have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamineN-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]Abuand P26D27[14−38]Abuand is probably native-like for N26G27K28[14−38]Abu. [R]Abualso shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.