Derivation of structural restraints using a thiol-reactive chelator

被引：61

作者：

Dvoretsky, A ^{[1
]}

Gaponenko, V ^{[1
]}

Rosevear, PR ^{[1
]}

机构：

[1] Univ Cincinnati, Coll Med, Dept Mol Genet Biochem & Microbiol, Cincinnati, OH 45267 USA

来源：

FEBS LETTERS | 2002年 / 528卷 / 1-3期

关键词：

NMR paramagnetic restraint; residual dipolar couplings dipolar shift; barnase; protein fold;

D O I：

10.1016/S0014-5793(02)03297-0

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：189 / 192

页数：4

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