Communication

Journal of Biomolecular NMR

, Volume 49, Issue 1, pp 3-7

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

  • Mary Anne RefaeiAffiliated withDepartment of Chemistry, University of CincinnatiDepartment of Biochemistry, The Ohio State University
  • , Al CombsAffiliated withDepartment of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati
  • , Douglas J. KojetinAffiliated withDepartment of Molecular Genetics, Biochemistry and Microbiology, University of CincinnatiDepartment of Molecular Therapeutics, The Scripps Research Institute—Scripps Florida
  • , John CavanaghAffiliated withDepartment of Molecular and Structural Biochemistry, North Carolina State University
  • , Carol CaperelliAffiliated withCollege of Pharmacy, University of Cincinnati
  • , Mark RanceAffiliated withDepartment of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati
  • , Jennifer SapitroAffiliated withDepartment of Chemistry, University of Cincinnati
  • , Pearl TsangAffiliated withDepartment of Chemistry, University of Cincinnati Email author 

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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.

Keywords

Segmental labeling Sortase A Protein ligation MecA Enzyme-mediated ligation NMR