Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor.
Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor.
复制标题
还原和未折叠的牛胰腺胰蛋白酶抑制剂中广泛的非随机结构。
DOI：
10.1021/bi00043a002
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Woodward,C
中科院分区：
文献类型：
--
作者：
Pan,H;Barbar,E;Barany,G;Woodward,C
1995年8月15日收到的修订手稿？摘要：指定了一种在pH 4.5，1℃条件下还原的BPTI类似物的二维~1H-核磁共振谱。光谱显示了相当大的构象平均化，正如预期的那样，柔性的未折叠的蛋白质。通过NH、-NH、-+I和芳香族脂肪族NO的存在，可以检测到广泛的非随机结构的存在。顺序的酰胺-酰胺NOE表明，在链上的18对残基上有显著的转弯构象。在天然BPTI中，其中许多片段位于转角、环状或螺旋中，但在由残基29-35组成的片段中，观察到6个连续的对，在天然BPTI中，残基构成了一条延伸的片断。对于未折叠蛋白质的一个新发现是我们观察到的NOE暗示了非天然的疏水相互作用。对于彼此在1-3个残基内的残基对，观察到多个芳香族-脂肪族NO.大多数是非天然的，涉及天然BPTI的中心反平行链-转折链的两条链中的残基，由18-35个残基组成。[Kemmink，J“&Creighton，T.(1993)J.Mol.比奥尔。234,861-878]在减少的BPTI中观察到，但也存在许多其他的。天然BPTI被二硫苏糖醇还原，半胱氨酸被α-氨基正丁酸取代，BPTI突变体F45A被二硫苏糖醇还原，得到了类似的光谱。许多转角状构象和疏水相互作用的迹象与早期的报告一致，即还原的BPTI是一种熔融卷曲，它在一定程度上坍塌，但不像天然的那样多，并且暴露出成簇的疏水分子[Ferrer，M.，Barany，G.，&Woodward，C.(1995)Natural Struct。比奥尔。2,211-217]，将还原BPTI中的NOE与早期BPTI折叠中间体模型中的NOE进行比较，表明非天然相互作用在BPTI折叠的初始步骤中起着重要作用。还原BPTI的一个变体，其中所有半胱氨酸都被5-[13C]甲基半胱氨酸取代，在HMQC检测到的[13C]甲基质子的共振中也显示出化学位移分散。加入盐酸胍后，分散性消失，表明变性剂破坏了还原BPTI中的非随机结构。
Revised Manuscript Received August 15, 1995® abstract: Two-dimensional'H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 C, have been assigned. Spectra indicate considerable conformational averaging, as expected for a flexible, unfolded protein. The presence of extensive nonrandom structure is detected by the presence of NH,—NH,-+ i and aromatic—aliphatic NOEs. Sequential amide—amide NOEs indicate that turn-like conformations are significantly populated at 18 pairs of residues along the chain. Many of these are located in a turn, loop, or helix in native BPTI, butsix are observed for contiguous pairs in the segment composed of residues 29—35, which in native BPTI constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic interactions. Multiple aromatic—aliphatic NOEs are observed for pairs of residues that are within 1—3 residues of each other. Most are non-native and involve residues in both strands of the central antiparallel strand—turn—strand of native BPTI comprised of residues 18—35. All NOEs reported for oligopeptides spanning the BPTI sequence [Kemmink, J „& Creighton, T.(1993) J. Mol. Biol. 234, 861—878] are observed in reduced BPTI, but manyothers are present as well. Similar spectra are obtained for naturally occurring BPTI reduced by dithiothreitol, BPTI with cysteines replaced by a-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol. The indications of numerous turn-like conformations and of hydrophobic interactions are consistent with earlier reports that reduced BPTI is a molten coil which is collapsed to some extent but not as much as native, and which has exposed, clustered hydrophobes [Ferrer, M., Barany, G., & Woodward, C.(1995) Nature Struct. Biol. 2, 211—217], Comparison of the NOEs in reduced BPTI to those in a model for early BPTI folding intermediates suggests a significant role for non-native interactions in initial steps ofBPTI folding. A variant of reduced BPTI, in which all cysteines are replaced with 5-[13C] methylcysteine, also displays chemicalshift dispersion in HMQC-detected resonances of the [13C] methyl protons. The dispersion is lost by addition of guanidine hydrochloride, indicating that nonrandom structure in reduced BPTI is disrupted by the denaturant.