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Published October 2015 | public
Journal Article Open

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications


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.

Additional Information

© 2015 American Chemical Society. Received: August 10, 2015; Revised: September 11, 2015; Publication Date (Web): October 2, 2015. We thank members of D.A.T.'s and T.K.U.'s research groups for helpful discussions. We are grateful to Prof. Stephen Mayo (Caltech) and Prof. Richard Kahn (Emory University) for their gifts of plasmids. The staff of the Proteome Exploration Laboratory of the Caltech Beckman Institute kindly assisted with mass spectrometry. Support for this work was provided by the Caltech Innovation Initiative (CI2), the Jacobs Institute for Molecular Engineering for Medicine, a Caltech Summer Undergraduate Research Fellowship (to M.L.), the Purdue Research Foundation, and a Purdue HHMI Summer Research Fellowship (to J.G.F.). The authors declare no competing financial interest.

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August 20, 2023
August 20, 2023