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

Academic Article
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Overview

authors

  • Scrofani, S. D. B.
  • Wright, Peter
  • Dyson, Jane

publication date

  • November 1998

journal

  • Protein Science  Journal

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 13Cd-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 1H spectral overlap. Here, we describe an alternative approach, where metal-binding ligands such as histidine and cysteine are specifically 15N backbone labeled, excess EDTA is added and changes to (1H-15N) HSQC spectra are followed. Under these conditions, the amide groups of all 15N 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.

subject areas

  • Bacteroides fragilis
  • Binding Sites
  • Cysteine
  • Edetic Acid
  • Histidine
  • Magnetic Resonance Spectroscopy
  • Molecular Weight
  • Nitrogen Isotopes
  • Recombinant Proteins
  • Zinc
  • beta-Lactamases
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Research

keywords

  • NMR
  • metal binding ligands
  • metallo-beta-lactamase
  • metalloprotein
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Identity

PubMed Central ID

  • PMC2143851

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1002/pro.5560071128

PubMed ID

  • 9828017
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Additional Document Info

start page

  • 2476

end page

  • 2479

volume

  • 7

issue

  • 11

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