title: Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
abstract: NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is approximately 40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati 45221-0172, USA.
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Refaei
authors.forename: Al
authors.initials: A
authors.lastname: Combs
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Kojetin
authors.forename: John
authors.initials: J
authors.lastname: Cavanagh
authors.forename: Carol
authors.initials: C
authors.lastname: Caperelli
authors.forename: Mark
authors.initials: M
authors.lastname: Rance
authors.forename: Jennifer
authors.initials: J
authors.lastname: Sapitro
authors.forename: Pearl
authors.initials: P
authors.lastname: Tsang
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D009587:Nitrogen Isotopes; D011506:Proteins; D011993:Recombinant Fusion Proteins; C083616:mecA protein, Bacillus subtilis; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases