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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 ~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.

• Content Type Journal Article
• DOI 10.1007/s10858-010-9464-2
• Authors
  • Mary Anne Refaei, Department of Chemistry, University of Cincinnati, Cincinnati, 45221-0172 USA
  • Al Combs, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, 45267-0524 USA
  • Douglas J. Kojetin, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, 45267-0524 USA
  • John Cavanagh, Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA
  • Carol Caperelli, College of Pharmacy, University of Cincinnati, Cincinnati, 45267-0004 USA
  • Mark Rance, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, 45267-0524 USA
  • Jennifer Sapitro, Department of Chemistry, University of Cincinnati, Cincinnati, 45221-0172 USA
  • Pearl Tsang, Department of Chemistry, University of Cincinnati, Cincinnati, 45221-0172 USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR