The identification of metal-binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy

The identification of metal-binding ligands in metalloproteins is an important step in gaining detailed information regarding the environment of the active site. Traditionally, techniques such as Cd-113-substitution for the active metal followed by isotope-filtered NMR techniques have been used to this end. However, for medium to high molecular weight proteins (>20 kDa), these experiments may not be beneficial due to extensive H-1 spectral overlap. Here, we describe an alternative approach, where metal-binding ligands such as histidine and cysteine are specifically N-15 backbone labeled, excess EDTA is added and changes to {H-1-N-15} HSQC spectra are followed. Under these conditions, the amide groups of all N-15 labeled histidine and cysteine residues, which were either ligands or residues close to the active site, were identified unambiguously for metallo-beta-lactamase from Bacteroides fragilis.