title: Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications.
abstract: Calmodulin (CaM) is a widely studied Ca(2+)-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca(2+) or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Blvd., Pasadena, California 91125, United States.
authors.forename: Chethana
authors.initials: C
authors.lastname: Kulkarni
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Blvd., Pasadena, California 91125, United States.
authors.forename: Megan
authors.initials: M
authors.lastname: Lo
authors.affiliation: Weldon School of Biomedical Engineering, Purdue University , 206 South Martin Jischke Drive, West Lafayette, Indiana 47907, United States.
authors.forename: Julia G
authors.initials: JG
authors.lastname: Fraseur
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Blvd., Pasadena, California 91125, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Blvd., Pasadena, California 91125, United States.|Weldon School of Biomedical Engineering, Purdue University , 206 South Martin Jischke Drive, West Lafayette, Indiana 47907, United States.
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Kinzer-Ursem
mesh_terms: D000217:Acyltransferases / Q000737:chemistry; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification; D002853:Chromatography, Liquid / Q000379:methods; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007850:Lauric Acids / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D012117:Resins, Synthetic / Q000737:chemistry
chemical_list: C000609689:12-azidododecanoic acid; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D007850:Lauric Acids; D012117:Resins, Synthetic; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D019703:Calcineurin; D002118:Calcium