Site-specific labelling with a metal chelator for protein-structure refinement

    loading  Checking for direct PDF access through Ovid

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. 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.

Related Topics

    loading  Loading Related Articles