PMID:15213433
| Citation |
Pintacuda, G, Moshref, A, Leonchiks, A, Sharipo, A and Otting, G (2004) Site-specific labelling with a metal chelator for protein-structure refinement. J. Biomol. NMR 29:351-61 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1023/B:JNMR.0000032610.17058.fe |
| Keywords |
Bacterial Proteins/chemistry; Chelating Agents/chemistry; Chelating Agents/pharmacology; Cobalt/chemistry; Copper/chemistry; Cysteine/chemistry; DNA/chemistry; Edetic Acid/chemistry; Electron Spin Resonance Spectroscopy; Electrons; Escherichia coli/metabolism; Gadolinium/chemistry; Ions; Magnetic Resonance Spectroscopy/methods; Manganese/chemistry; Models, Chemical; Models, Molecular; Protein Structure, Tertiary; Proteins/chemistry; Protons; Repressor Proteins/chemistry; Time Factors |
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