Pintacuda, GuidoMoshref, AhmadLeonchiks, AinarsSharipo, AnatolyOtting, Gottfried2015-12-132015-12-130925-2738http://hdl.handle.net/1885/78459A single free Cys sidechain in the N-terminal domain of the E. coli arginine repressor was covalently derivatized with S-cysteaminyl-EDTA for site-specific attachment of paramagnetic metal ions. The effects of chelated metal ions were monitored with 15N-HSQC spectra. Complexation of Co2+, which has a fast relaxing electron spin, resulted in significant pseudocontact shifts, but also in peak doubling which was attributed to the possibility of forming two different stereoisomers of the EDTA-Co2+ complex. In contrast, complexation of Cu2+ or Mn2+, which have slowly relaxing electron spins, did not produce chemical shift changes and yielded self-consistent sets of paramagnetic relaxation enhancements of the amide protons. T1 relaxation enhancements with Cu2+ combined with T2 relaxation enhancements with Mn2+ are shown to provide accurate distance restraints ranging from 9 to 25 Å. These long-range distance restraints can be used for structural studies inaccessible to NOEs. As an example, the structure of a solvent-exposed loop in the N-terminal domain of the E. coli arginine repressor was refined by paramagnetic restraints. Electronic correlation times of Cu2+ and Mn2+ were determined from a comparison of T1 and T2 relaxation enhancements.Keywords: arginine; chelating agent; cobalt; copper ion; cysteine; edetic acid derivative; manganese; metal chelate; repressor protein; amino terminal sequence; article; calculation; complex formation; controlled study; correlation analysis; covalent bond; electron Inversion-recovery; Paramagnetic relaxation enhancement; Paramagnetic restraints; Protein derivatization; S-cysteaminyl-EDTA200410.1023/B:JNMR.0000032610.17058.fe2015-12-11