Derivation of structural restraints using a thiol‐reactive chelator

FEBS Letters

Gaponenko, Vadim¹  Rosevear, Paul R¹  Dvoretsky, Alex¹ 

[1]Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267, USA

关键词: NMR;    Paramagnetic restraint;    Residual dipolar coupling;    Dipolar shift;    Barnase;    Protein fold;    thiol-reactive EDTA;    S-(2-pyridylthio)cysteaminyl ethylenediamine tetraacetic acid;    barnase(H102C-EDTA);    barnase with histidine 102 mutated to cysteine and modified with S-(2-pyridylthio)cysteaminyl-ethylenediaminetetraacetic acid;    barnase(H102A);    barnase with histidine 102 mutated to alanine;    α/β TROSY;    α/β transverse relaxation optimized spectroscopy;    RDC;    residual dipolar coupling;    DCS;    dipolar chemical shift;    NMR;    nuclear magnetic resonance;    rms;    root mean square;   

DOI  :  10.1016/S0014-5793(02)03297-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 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.

【 授权许可】

Unknown