Resonance Assignments of Lowly Populated and Unstable Enzyme Intermediate Complex under Real-Time Conditions

Chembiochem. 2019 Nov 4;20(21):2738-2742. doi: 10.1002/cbic.201900240. Epub 2019 Oct 1.

Abstract

Unstable and low-abundance protein complexes represent a large family of transient protein complexes that are difficult to characterize, even by means of high-resolution NMR spectroscopy. A method to assign the NMR signals of these unstable complexes through a combination of selective isotope labeling of amino acids in a protein and site-specific labeling the protein with a paramagnetic tag is presented herein. By using this method, the resonances of unstable thioester intermediate complex (lifetime <5 h and highest concentration ≈20 μm) generated by Staphylococcus aureus sortase A and its peptide substrate under a real-time reaction have been assigned.

Keywords: NMR spectroscopy; enzymes; isotope effects; proteins; real-time reactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry*
  • Amino Acids / metabolism
  • Aminoacyltransferases / chemistry
  • Aminoacyltransferases / genetics
  • Aminoacyltransferases / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • Enzyme Stability
  • Isotope Labeling / methods*
  • Models, Molecular
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism
  • Nitrogen Isotopes / chemistry
  • Nitrogen Isotopes / metabolism
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Protein Binding
  • Protein Conformation*
  • Staphylococcus aureus / enzymology
  • Staphylococcus aureus / genetics
  • Substrate Specificity

Substances

  • Amino Acids
  • Bacterial Proteins
  • Multienzyme Complexes
  • Nitrogen Isotopes
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases