Protein Science, Vol 7, Issue 11 2476-2479, Copyright © 1998 by Cold Spring Harbor Laboratory Press

FOR THE RECORD

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

SDB. SCROFANI, P. E. WRIGHT and H. J. DYSON
Present address: AMRAD Operations, Private Box 29, Richmond, Victoria, 3121, Australia.

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 (113)Cd-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 (1)H spectral overlap. Here, we describe an alternative approach, where metal-binding ligands such as histidine and cysteine are specifically (15)N backbone labeled, excess EDTA is added and changes to {(1)H-(15)N} HSQC spectra are followed. Under these conditions, the amide groups of all (15)N 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.
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