Derivation of structural restraints using a thiol-reactive chelator

Alex Dvoretsky, Vadim Gaponenko, Paul R Rosevear
2002 FEBS Letters  
Recognition and identi¢cation 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 modi¢cation 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 Yb 3+ , Mn 2+ , and Co 2+ permitted
more » ... 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 su⁄cient for validation of homology-based fold predictions. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
doi:10.1016/s0014-5793(02)03297-0 pmid:12297302 fatcat:2vlw6xh36zahhgk2stga3jg2ka