Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy.
Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy.
复制标题
通过同核和异核核磁共振波谱重新检查大肠杆菌含组氨酸蛋白质的二级和三级结构。
DOI：
10.1021/bi00115a014
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Klevit,RE
中科院分区：
文献类型：
--
作者：
Hammen,PK;Waygood,EB;Klevit,RE
修订稿于 1991 年 10 月 16 日收到 摘要：通过二维同核和异核核磁共振技术对大肠杆菌中的含组氨酸蛋白 (HPr) 进行了分析，扩展了最初报道的工作 [Klevit, R. E., Drobny, G. D., & Waygood, EB (1986) Biochemistry 25, 7760-7769; Klevit, R. E. 和 Drobny, G. P.(1986) 生物化学 25, 7770-7773； Klevit, R. E. 和 Waygood, EB (1986) 生物化学 25, 7774-7781]。二维同核全相干光谱（TOCSY）可以比原始研究中更完整地分配侧链自旋系统。此外，二维 15N-* H 异核光谱用于解决同核光谱中由于共振冗余而存在的许多模糊性。这些分析修正了许多共振分配，这些共振分配是用 6 年前存在的技术可以收集的光谱进行的。此外，还测量了酰胺交换率和 3/NH 偶联常数，扩展了原始分析并产生了新的结构信息。所有这些数据都被用来重新检查大肠杆菌 HPr 的折叠拓扑。结构计算表明，从早期 NMR 数据得出的拓扑结构，即四链/三片，其中三个 a 螺旋沿着片的一侧延伸，基本上没有变化，尽管在目前的分析水平上，无法以高置信度建立明确定义的“螺旋 B”。此外，这里报告的数据揭示了属于 Ser31 和 Thr59 的两个缓慢交换的侧链羟基质子的存在。它们的行为强烈表明这些侧链涉及氢键。这两个残基均位于 3-折叠的边缘，可能有助于该结构的极端稳定性。五年前，报道了来自大肠杆菌的含组氨酸蛋白 (HPr) 的折叠拓扑结构 (Klevit & Waygood，1986)。基于二维同核核磁共振谱的定性分析，我们得出结论，85个残基的磷酸转移蛋白由四链反平行/8片组成，其中三个α螺旋与片大致平行且彼此反平行。在该报告之后，对来自革兰氏阳性细菌枯草芽孢杆菌的相关 HPr 进行了类似的分析（Wittekind 等人，1990）。 NMR 数据再次表明存在四链反平行/8 片以及大肠杆菌蛋白质中所示的三个 α 螺旋中的两个。然而，从大肠杆菌 HPr 的 X 射线衍射研究中得出的三级结构与 NMR 结果表明的拓扑结构显着不同，由两条彼此大致平行的双链/8 片层和两个垂直于这些片层延伸的 α 螺旋组成（El-Kabbani 等，1987）。为了解决 NMR 和 X 射线结构之间的明显差异，我们着手从 NMR 数据中确定 HPr 的详细结构。作为该过程的一部分，额外的 'H-'H 同核和 15N-'H 异核
Revised Manuscript Received October 16, 1991 abstract: Analysis of the histidine-containing protein (HPr) fromEscherichia coli by two-dimensional homonuclear and heteronuclear nuclear magnetic resonance techniques has been performed, extending the work originally reported [Klevit, R. E., Drobny, G. D., & Waygood, EB (1986) Biochemistry 25, 7760-7769; Klevit, R. E., & Drobny, G. P.(1986) Biochemistry 25, 7770-7773; Klevit, R. E., & Waygood, EB (1986) Biochemistry 25, 7774-7781]. Two-dimensional homonuclear total coherence spectroscopy (TOCSY) allowed for more complete assignments of the side-chain spin systems than had been possible in the original studies. As well, two-dimensional 15N-* H heteronuclear spectroscopy was used to resolve a number of ambiguities present in the homonuclear spectra due to resonance redundancies. These analyses led to the correction of a number of resonance assignments that were made with the spectra that could be collected with the technology that existed 6 years ago. In addition, amide exchange rates and 3/NH coupling constantshave been measured, extending the original analysis and yielding new structural information. All these data have been used to reexamine the folding topology of E. coli HPr. Structure calculations showed that the topology derived from the earlier NMR data, ie, a four-stranded/3-sheet with three a-helices running along one side of the sheet, was essentially unchanged, although at the present level of analysis, a well-defined “helix B” could not be established with high confidence. In addition, the data reported here revealed theexistence of two slowly-exchanging side-chain hydroxyl protons belonging to Ser31 and Thr59. Their behavior strongly suggests that these side chains are involved in hydrogen bonds. These two residues are both at the edges of the/3-sheet and may contribute to the extreme stability of this structure.Five years ago the folding topology for the histidine-con-taining protein (HPr) from Escherichia coli was reported (Klevit & Waygood, 1986). On thebasis of a qualitative analysis of two-dimensional homonuclear'Hnoesy1 NMR spectra, we concluded that the 85-residue phosphotransfer protein consists of a four-stranded antiparallel/8-sheet, with three a-helices running approximately parallel to the sheet and antiparallel to each other. Subsequent to that report, a similar analysis was performed for a related HPr from the Grampositive bacterium Bacillus subtilis (Wittekind et al., 1990). Again, the NMR data indicated the presence of a four-strand antiparallel/8-sheet as well as two of the three a-helices in-dicated in the E. coli protein. However, the tertiary structure derived from X-ray diffraction studies on E. coli HPr was significantly different from the topology suggested by the NMR results, consisting of two two-stranded/8-sheets that are approximately parallel to each other and two a-helices that run perpendicular tothese sheets (El-Kabbani et al., 1987). In an attempt toaddress the apparent discrepancy between the NMR and X-ray structures, we set out to determine a detailed structure forHPr from NMR data. As part of this process, additional'H-'H homonuclear and 15N-'H hetero-