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 (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.
Pintacuda
Guido
G
Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
Moshref
Ahmad
A
Leonchiks
Ainars
A
Sharipo
Anatoly
A
Otting
Gottfried
G
D001426:Bacterial Proteins / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology*; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D005682:Gadolinium / Q000737:chemistry; D007477:Ions; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008345:Manganese / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D012097:Repressor Proteins / Q000737:chemistry; D013997:Time Factors
C087018:ArgR protein, Bacteria; D001426:Bacterial Proteins; D002614:Chelating Agents; D007477:Ions; D011506:Proteins; D011522:Protons; D012097:Repressor Proteins; D003035:Cobalt; D008345:Manganese; D003300:Copper; D004247:DNA; D004492:Edetic Acid; D005682:Gadolinium; D003545:Cysteine