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

Investigating Activity of Engineered Proteins on Inorganic Substrates for Downstream Biomedical Applications

As the proteomics field continues to expand for biomedical research purposes, scientists are looking to integrate cross-disciplinary tools for studying protein structures, functions, and interactions. One protein of interest, calmodulin (CaM), is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes. To facilitate biophysical studies of calmodulin, researchers have designed site-specific labeling processes for use in bioconjugation applications while maintaining high levels of protein activity. Here, I present a platform for the highly efficient and selective immobilization of calmodulin onto different surfaces using azide-alkyne cycloaddition “click” chemistries. In particular, chemoenzymatically modified calmodulin is conjugated to sepharose resin beads for use in a novel affinity purification technique. I then present a method for in situ conjugation of calmodulin onto gold nanoparticles with consistent orientation and high levels activity. Protein Bioconjugation directly from clarified lysates represents a significant advance in what is normally a multi-step conjugation process. We anticipate these methods to be translatable to other proteins of interest and used in other downstream protein characterization assays

Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification.

As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this "next-generation" calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics

CaMKII purification.

<p>Representative SDS-PAGE of Ca²⁺/calmodulin-dependent kinase II (CaMKII) purified with two different types of affinity resins, comparing both quantity and quality of pure result. CaMKII appears at 50 kDa and is of most interest in this figure. A) Tradition methods using His-tagged affinity Ni-NTA resin B) our affinity resin generated by functionalizing NHS Sepharose with 12-ADA labeled CaM from cell lysate. CL = clarified lysate, E = elution.</p

Calcineurin (CaN) purification with various CaM resins.

<p>(A) Representative SDS-PAGE gels with Coomassie stain from CaN purifications done in parallel. Calmodulin Sephaorse 4B from GE (GE) is compared to 12-ADA CaM resins made with 1, 2, and 5 mg mL^-1 purified 12-ADA CaM. Both the α and β subunits of CaN appear at the correct MW (60 and 19 kDa, respectively). (B) Average total mg of CaN purified for each resin (n≥3). Repeats of Calmodulin Sepharose 4B were from different lots. Repeats of each resin made with different concentrations of 12-ADA CaM were made with different lots of DBCO-PEG₄-amine and NHS-Sepharose. Higher quantities of CaN indicate better resin performance and thus reflect ideal CaM concentration. GE = commercial standard Calmodulin Sepharose 4B, 1mg mL^-1 = resin prepared with 1 mg mL^-1 pure 12-ADA CaM, 5 mg mL^-1 = resin prepared with 5 mg mL^-1 pure 12-ADA CaM, Lysate = resin prepared with 12-ADA CaM straight from crude cell lysate, W = wash, E = elution. (C) Comparison of resins made with click chemistry functional groups at different concentrations (n≥3). Total average amount of 12-ADA CaM bound to the resins as calculated by measuring the concentration of 12-ADA CaM in solution before and after conjugation. DBCO is resin prepared with DBCO-PEG₄-amine. Alkyne are resins prepared with alkyne-PEG₄-amine. Quenched is resin that was incubated with 1M Tris to deactivate NHS-esters instead of a click chemistry functional group. * = p≤0.05, ** = p≤0.01.</p

Average amount of CaN purified from CaM resins.

<p>Average amount of CaN purified from CaM resins.</p

Quantification of peptides cleaved from CaM affinity resins.

<p>Trypsin digestion of the resins followed by colormetric quantification indicates that more peptide was cleaved from the next generation CaM affinity resin generated by conjugation of 12-ADA CaM from clarified lysate than from the commercially available CaM affinity resin (GE CaM Seph). * = p≤0.05 n = 3.</p

Reagents used in this study.

<p><b>(A)</b> Click chemistry reagents used in this study. <b>1.</b> 12-azidododecanoic acid (12-ADA), <b>2.</b> dibenzocyclooctyne-(polyethylene glycol)₄-amine (DBCO-PEG₄-amine), <b>3.</b> alkyne-(polyethylene glycol)₄-amine (alkyne-PEG₄-amine). <b>(B)</b> calmodulin rendered as a ribbon structure with 12-ADA covalently attached to the amino-terminus (not to scale). PDB ID = 1CLL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197120#pone.0197120.ref014" target="_blank">14</a>].</p

Production and use of next generation CaM resin.

<p>Overview of the production of our next generation CaM affinity resin and CaM-binding protein purification. Schematic shows the entire process sequentially. 1. Recombinant protein expression and co-translational labeling of engineered CaM with 12-ADA (terminal azide group denoted with N₃ stars). 2. Incubation of clarified lysate containing the 12-ADA CaM with DBCO-functionalized Sepharose resin (black spheres) where 12-ADA CaM covalently conjugates to the resin. 3. Washed next generation CaM affinity resin is incubated with a crude mixture containing the CaM-binding protein to be purified. 4. After washing and elution from the resin, purified CaM-binding protein is obtained.</p