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pubmed:abstractText |
A 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 (15)N-HSQC spectra. Complexation of Co(2+), 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-Co(2+) complex. In contrast, complexation of Cu(2+) or Mn(2+), 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. T (1) relaxation enhancements with Cu(2+) combined with T (2) relaxation enhancements with Mn(2+) are shown to provide accurate distance restraints ranging from 9 to 25 A. 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 Cu(2+) and Mn(2+) were determined from a comparison of T (1) and T (2) relaxation enhancements.
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