STUDIES ON THE SOLUTION CONFORMATION OF HUMAN THIOREDOXIN USING HETERONUCLEAR N-15-H-1 NUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY
STUDIES ON THE SOLUTION CONFORMATION OF HUMAN THIOREDOXIN USING HETERONUCLEAR N-15-H-1 NUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY
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DOI：
10.1021/bi00458a030
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发表时间：
1990-02-13
期刊：
影响因子：
2.9
通讯作者：
CLORE, GM
中科院分区：
文献类型：
--
作者：
FORMANKAY, JD;GRONENBORN, AM;CLORE, GM
本文用二维核磁共振方法研究了均匀标记的15N人硫氧还蛋白的溶液构象。蛋白质的15N共振的光谱以顺序的方式使用异质多重量子相干（HMQC）、中继HMQC相关（COSY）和中继HMQC-核Overhauser（NOESY）光谱获得。3JHN α的值从HMQC-COSY实验的变体（称为HMQC-J）中提取硫氧还蛋白的105个残基中的87个残基的裂解物，并分析以得到准确的3JHN α。耦合常数此外，通过异质多重键相关（HMBC）光谱鉴定长程C α H（i）-15 N（i +1）标量连接性。这些三键标量连接性主要存在于α-螺旋区与先前从人的细胞核Overhauser数据的定性分析中确定的二级结构很好地相关[Forman-Day，J.D.，克洛尔，G。M.，Drivel，P. C.，温菲尔德，P.T.，理查兹，F. M.，和Gronenborn，A.M.（1989）Biochemistry 28，7088 - 7097]，表明该技术可以为先验的二级结构确定提供额外的信息。3JHN α.偶联常数可以从HMQC-J实验中获得，从而可以更精确地描绘人硫氧还蛋白二级结构元件的起始和末端以及这些元件中的不规则性。
The solution conformation of uniformly labeled 15N human thioredoxin has been studied by two-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Assignments of the 15N resonances of the protein are obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC-correlated (COSY), and relayed HMQC-nuclear Overhauser (NOESY) spectroscopy. Values of the 3JHN.alpha. splittings for 87 of the 105 residues of thioredoxin are extracted from a variant of the HMQC-COSY experiment, known as HMQC-J, and analyzed to give accurate 3JHN.alpha. coupling constants. In addition, long-range C.alpha.H(i)-15N(i+1) scalar connectivities are identified by heteronuclear multiple bond correlation (HMBC) spectroscopy. The presence of these three-bond scalar connectivities in predominantly .alpha.-helical regions correlates well with the secondary structure determined previously from a qualitative analysis of homonuclear nuclear Overhauser data [Forman-Day, J.D., Clore, G. M., Driscoll, P. C., Wingfield, P.T., Richards, F. M., and Gronenborn, A.M. (1989) Biochemistry 28, 7088-7097], suggesting that this technique may provide additional information for secondary structure determination a priori. The accuracy with which 3JHN.alpha. coupling constants can be obtained from the HMQC-J experiment permits a more precise delineation of the beginnings and ends of secondary structural elements of human thioredoxin and of irregularities in these elements.