期刊
JOURNAL OF BIOMOLECULAR NMR
卷 29, 期 3, 页码 351-361出版社
SPRINGER
DOI: 10.1023/B:JNMR.0000032610.17058.fe
关键词
inversion-recovery; paramagnetic relaxation enhancement; paramagnetic restraints; protein derivatization; S-cysteaminyl-EDTA
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 N-15-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. T-1 relaxation enhancements with Cu2+ combined with T-2 relaxation enhancements with Mn2+ are shown to provide accurate distance restraints ranging from 9 to 25 Angstrom. 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 T-1 and T-2 relaxation enhancements.
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