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

✍ Scribed by Sergiod. B. Scrofani; Peter E. Wright; H. Jane Dyson

• Book ID: 105356572
• Publisher: Cold Spring Harbor Laboratory Press
• Year: 1998
• Tongue: English
• Weight: 333 KB
• Volume: 7
• Category: Article
• ISSN: 0961-8368
• DOI: 10.1002/pro.5560071128

✦ Synopsis

Abstract

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 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 {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‐ß‐lactamse from Bacteroides fragilis.