bc5b00449_si_001.pdf (2.49 MB)
Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications
journal contribution
posted on 2015-10-21, 00:00 authored by Chethana Kulkarni, Megan Lo, Julia G. Fraseur, David
A. Tirrell, Tamara L. Kinzer-UrsemCalmodulin (CaM) is a widely studied
Ca2+-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
Ca2+ 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.