Derivation of structural restraints using a thiol-reactive chelator.
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 Yb(3+), Mn(2+), and Co(2+) 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.
Dvoretsky
Alex
A
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, OH 45267, USA.
Gaponenko
Vadim
V
Rosevear
Paul R
PR
D001426:Bacterial Proteins; D001665:Binding Sites; D002614:Chelating Agents; D003545:Cysteine / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives*; D008670:Metals / Q000737:chemistry; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D001426:Bacterial Proteins; D002614:Chelating Agents; D008670:Metals; D011506:Proteins; C098697:S-(2-nitrophenylsulfenyl)cysteaminyl EDTA; D013438:Sulfhydryl Compounds; D004492:Edetic Acid; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D003545:Cysteine