Journal
FEBS LETTERS
Volume 528, Issue 1-3, Pages 189-192Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0014-5793(02)03297-0
Keywords
NMR paramagnetic restraint; residual dipolar couplings dipolar shift; barnase; protein fold
Funding
- NIAMS NIH HHS [AR 44324] Funding Source: Medline
- NIGMS NIH HHS [GM 62153] Funding Source: Medline
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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.
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