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

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.