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The identification of metal‐binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy
Author(s) -
Scrofani Sergiod. B.,
Wright Peter E.,
Dyson H. Jane
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560071128
Subject(s) - metalloprotein , nuclear magnetic resonance spectroscopy , nuclear magnetic resonance , chemistry , spectroscopy , metal , ligand (biochemistry) , physics , biochemistry , receptor , organic chemistry , quantum mechanics
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.