TWO-DIMENSIONAL H-1-NMR STUDY OF HUMAN UBIQUITIN - A MAIN CHAIN DIRECTED ASSIGNMENT AND STRUCTURE-ANALYSIS
TWO-DIMENSIONAL H-1-NMR STUDY OF HUMAN UBIQUITIN - A MAIN CHAIN DIRECTED ASSIGNMENT AND STRUCTURE-ANALYSIS
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DOI：
10.1021/bi00397a012
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发表时间：
1987-11-17
期刊：
影响因子：
2.9
通讯作者：
WAND, AJ
中科院分区：
文献类型：
--
作者：
DISTEFANO, DL;WAND, AJ
修订稿于 1987 年 7 月 6 日收到摘要：通过二维核磁共振技术研究了人类泛素的共振。应用了最近引入的分配算法，称为主链定向 (MCD) 分配 [Englander, S. W., & Wand, A. J.(1987) Biochemistry 26, 5953-5958]。该方法依赖于在二维/相关和核奥弗豪瑟效应谱中对指定连接模式的有序搜索，并以涉及主链酰胺 NH、-CH 和/3-CH 的偶极相互作用为中心。与顺序分配程序不同，MCD 方法不依赖于侧链/耦合网络的定义，并且通常不按蛋白质的一级序列顺序。各种MCD模式和通用算法被重申并应用于人类泛素的分析。通过该算法，绝大多数氨基酸残基酰胺NH-CqH-C^H/-偶联的亚自旋系统可以与二级结构单元关联并在其内对齐，而无需了解侧链的身份。通过限制侧链自旋系统的身份，极大地简化了对侧链自旋系统的识别。给出了基本上完整的共振分配。对 MCD 方法与顺序分配方法进行了一些详细的比较。 MCD 方法非常适合自动化。研究发现，在 pH* 5.8 和 30°C 下，人类泛素由涉及 5 条链的广泛/3 片结构组成。这些链中的三个形成共享公共链的反向平行组，并且具有与两个反向平行链平行的方向。还观察到两个螺旋段。最大的跨度为 13 个残基，显示出与 α 螺旋一致的偶极相互作用，而根据观察到的核奥弗豪瑟效应，较小的 4 残基螺旋段似乎是 310 螺旋。可以证明五个经典的急转。二维核磁共振技术现已导致许多小蛋白质的几乎完整的共振分配。小蛋白质中质子共振的分配通常通过应用 Wüthrich 及其同事引入和开发的直接顺序分配方法来进行
Revised Manuscript Received July 6, 1987 abstract: The resonances of human ubiquitin were studied by two-dimensional nuclear magnetic resonance techniques. A recently introduced assignment algorithm termed the main chain directed (MCD) assignment [Englander, S. W., & Wand, A. J.(1987) Biochemistry 26, 5953-5958] was applied. This approach relies on an ordered series of searches for prescribed patterns of connectivities in two-dimensional/-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH,-CH, and/3-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain/-coupled networks and is generally not sequential with the primary sequence of the protein. The various MCD patterns and the general algorithm are reiterated and applied to the analysis of human ubiquitin. With this algorithm, the vast majority of amino acid residue amide NH-CqH-C^ H/-coupled subspin systems could be associated with and aligned within units of secondary structure without any knowledge of the identity of the side chains. This greatly simplified recognition of side-chain spin systems by restricting their identity. Essentially complete resonance assignments are presented. The MCD method is compared with the sequential assignment method in some detail. The MCD method is highly amenable to automation. Human ubiquitin is found, at pH* 5.8 and 30 C, to be composed of an extensive/3-sheet structure involving five strands. Three of these strands form an antiparallel set sharing a common strand and have a parallel orientation to two antiparallel strands. Two helical segments were also observed. The largest, spanning 13 residues, shows dipolar interactionsconsistent with an-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 310 helix. Five classical tight turns could be demonstrated.Two-dimensional nuclear magnetic resonance techniques have now led to the nearly complete resonance assignments of a number of small proteins. The assignment of proton resonances in small proteins has usually proceeded by the application of the direct sequential assignment method introduced and developed by Wüthrich and co-workers