Di Stefano D L, Wand A J
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Biochemistry. 1987 Nov 17;26(23):7272-81. doi: 10.1021/bi00397a012.
The 1H 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 J-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH, alpha-CH, and beta-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain J-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-C alpha H-C beta H J-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 degrees C, to be composed of an extensive beta-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 interactions consistent with an alpha-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 3(10) helix. Five classical tight turns could be demonstrated.
采用二维核磁共振技术研究了人泛素的1H共振。应用了一种最近引入的称为主链导向（MCD）归属的算法[英格兰德，S.W.，& 万德，A.J.（1987年）《生物化学》26卷，5953 - 5958页]。该方法依赖于在二维J相关谱和核Overhauser效应谱中对规定连接模式进行有序的一系列搜索，并以涉及主链酰胺NH、α-CH和β-CH的偶极相互作用为核心。与顺序归属程序不同，MCD方法不依赖于侧链J耦合网络的定义，并且通常与蛋白质的一级序列不具有顺序性。反复阐述了各种MCD模式和通用算法，并将其应用于人泛素的分析。使用该算法，在不了解侧链身份的情况下，绝大多数氨基酸残基的酰胺NH-CαH-CβH J耦合亚自旋系统能够与二级结构单元相关联并排列在其中。这通过限制侧链自旋系统的身份极大地简化了对它们的识别。给出了基本完整的共振归属。将MCD方法与顺序归属方法进行了较为详细的比较。MCD方法非常适合自动化。发现在pH 5.8和30℃条件下，人泛素由一个涉及五条链的广泛β折叠结构组成。其中三条链形成一个反平行组，共享一条公共链，并与两条反平行链呈平行取向。还观察到两个螺旋段。最大的螺旋段跨越13个残基，显示出与α螺旋一致的偶极相互作用，而较小的4残基螺旋段根据观察到的核Overhauser效应似乎是一个3(10)螺旋。可以证明有五个典型的紧密转角。
