Derivation of structural restraints using a thiol-reactive chelator
Derivation of structural restraints using a thiol-reactive chelator
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DOI：
10.1016/s0014-5793(02)03297-0
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发表时间：
2002-09-25
期刊：
影响因子：
3.5
通讯作者：
Rosevear, PR
中科院分区：
文献类型：
--
作者：
Dvoretsky, A;Gaponenko, V;Rosevear, PR
蛋白质折叠的识别和鉴定是高通量结构基因组学的先决条件。在这里，我们展示了一种简单的方案，通过化学修饰的单一半胱氨酸残基在麻秆酶（H102C）中共价附着一个短而更刚性的金属螯合标签，巯基反应EDTA。金属螯合标签的共轭提供了允许更大范围的顺磁性金属取代的优点。取代Yb3+， Mn2+和Co2+允许测量金属-酰胺质子距离，偶极位移和残余偶极耦合。顺磁衍生约束在大型蛋白质复合物的核磁共振结构解析中是有利的，并且足以验证基于同源性的折叠预测。(C) 2002年由Elsevier Science B.V.代表欧洲生化学会联合会出版。
Recognition and identification of protein folds is a prerequisite for high-throughput structural genomics. Here we demonstrate a simple protocol for covalent attachment of a short and more rigid metal-chelating tag, thiol-reactive EDTA, by chemical modification of the single cysteine residue in barnase(H102C). Conjugation of the metal-chelating tag provides the advantage of allowing a greater range of paramagnetic metal substitutions. Substitution of Yb3+, Mn2+, and Co2+ permitted measurement of metal-amide proton distances, dipolar shifts, and residual dipolar couplings. Paramagnetic-derived restraints are advantageous in the NMR structure elucidation of large protein complexes and are shown sufficient for validation of homology-based fold predictions. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.