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Site-specific Labelling with a Metal Chelator for Protein-structure Refinement

Journal of Biomolecular NMR volume 29, pages 351–361 (2004)Cite this article

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 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. 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 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 T 1 and T 2 relaxation enhancements.

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Affiliations

  1. Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171, Stockholm, Sweden

    Guido Pintacuda & Ahmad Moshref

  2. Biomedical Research and Study Centre, University of Latvia, LV-1067, Riga, Latvia

    Ainars Leonchiks & Anatoly Sharipo

  3. Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia

    Gottfried Otting

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  1. Guido Pintacuda
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  2. Ahmad Moshref
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  4. Anatoly Sharipo
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  5. Gottfried Otting
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Pintacuda, G., Moshref, A., Leonchiks, A. et al. Site-specific Labelling with a Metal Chelator for Protein-structure Refinement. J Biomol NMR 29, 351–361 (2004). https://doi.org/10.1023/B:JNMR.0000032610.17058.fe

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  • DOI: https://doi.org/10.1023/B:JNMR.0000032610.17058.fe

  • inversion-recovery
  • paramagnetic relaxation enhancement
  • paramagnetic restraints
  • protein derivatization
  • S-cysteaminyl-EDTA