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.
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acids / chemistry*
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Amino Acids / metabolism
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Aminoacyltransferases / chemistry
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Aminoacyltransferases / genetics
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Aminoacyltransferases / metabolism
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Cysteine Endopeptidases / chemistry
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / metabolism
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Enzyme Stability
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Isotope Labeling / methods*
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Models, Molecular
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Multienzyme Complexes / chemistry*
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Multienzyme Complexes / metabolism
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Nitrogen Isotopes / chemistry
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Nitrogen Isotopes / metabolism
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Nuclear Magnetic Resonance, Biomolecular / methods*
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Protein Binding
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Protein Conformation*
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Staphylococcus aureus / enzymology
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Staphylococcus aureus / genetics
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Substrate Specificity
Substances
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Amino Acids
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Bacterial Proteins
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Multienzyme Complexes
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Nitrogen Isotopes
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Aminoacyltransferases
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sortase A
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Cysteine Endopeptidases