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

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

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

Competition for CaM alters binding dynamics.

<p>Time-course of CaM binding partners bound to various states of CaM for 1 second of 10 Hz Ca²⁺ flux: CaM₀ (blue), CaM_2N (red), CaM_2C (green), CaM₄ (purple), and CaM_tot (orange). CaM_tot is the sum of all CaM-bound states for a given protein. The concentration of each species is normalized against its maximum value of CaM_tot. Solid lines denote the isolated model. Dotted lines denote the competitive model. The differences between isolated and competitive behavior are more significant for some CaM binding partners than others.</p

Schematic of CaM binding.

<p>(A) Structure of CaM (PDB 1CLL), shown in blue, with two Ca²⁺ ions (gold) at each terminus. (B) Structure of Ca²⁺/CaM (PDB 2JZI) bound to a calcineurin (CaN) peptide (red). (C) Schematic of CaM interactions with downstream binding partners. CaM may bind Ng in the absence of Ca²⁺. In the presence of Ca²⁺, CaM binds to CaN, CaMKII, NOS, MLCK, and AC1 and AC8.</p

Competitive tuning: Competition's role in setting the frequency-dependence of Ca²⁺-dependent proteins

<div><p>A number of neurological disorders arise from perturbations in biochemical signaling and protein complex formation within neurons. Normally, proteins form networks that when activated produce persistent changes in a synapse’s molecular composition. In hippocampal neurons, calcium ion (Ca²⁺) flux through N-methyl-D-aspartate (NMDA) receptors activates Ca²⁺/calmodulin signal transduction networks that either increase or decrease the strength of the neuronal synapse, phenomena known as long-term potentiation (LTP) or long-term depression (LTD), respectively. The calcium-sensor calmodulin (CaM) acts as a common activator of the networks responsible for both LTP and LTD. This is possible, in part, because CaM binding proteins are “tuned” to different Ca²⁺ flux signals by their unique binding and activation dynamics. Computational modeling is used to describe the binding and activation dynamics of Ca²⁺/CaM signal transduction and can be used to guide focused experimental studies. Although CaM binds over 100 proteins, practical limitations cause many models to include only one or two CaM-activated proteins. In this work, we view Ca²⁺/CaM as a limiting resource in the signal transduction pathway owing to its low abundance relative to its binding partners. With this view, we investigate the effect of competitive binding on the dynamics of CaM binding partner activation. Using an explicit model of Ca²⁺, CaM, and seven highly-expressed hippocampal CaM binding proteins, we find that competition for CaM binding serves as a tuning mechanism: the presence of competitors shifts and sharpens the Ca²⁺ frequency-dependence of CaM binding proteins. Notably, we find that simulated competition may be sufficient to recreate the <i>in vivo</i> frequency dependence of the CaM-dependent phosphatase calcineurin. Additionally, competition alone (without feedback mechanisms or spatial parameters) could replicate counter-intuitive experimental observations of decreased activation of Ca²⁺/CaM-dependent protein kinase II in knockout models of neurogranin. We conclude that competitive tuning could be an important dynamic process underlying synaptic plasticity.</p></div

Competitive tuning explains intermolecular crosstalk.

<p>(A) Simulations of CaMKII phosphorylation in our isolated model with and without inclusion of Ng. (B) Simulations of CaMKII phosphorylation in our competitive model with and without Ng. (C) CaMKII activity in WT and Ng^-/- knockout mice from Krucker <i>et al</i>. Simulations were performed to replicate the experimental method of Krucker <i>et al</i>. as closely as possible. (D) The average bound concentration (C_b) of each CaM binding protein in semi-isolated models as a function of Ng concentration. AC8-Ct and AC8-Nt exhibit the greatest relative change in CaM-binding (C_b, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005820#pcbi.1005820.e001" target="_blank">Eq 1</a>) as Ng concentration decreases. (E) The average bound concentration (C_b) of each CaM binding protein in the competitive model as a function of Ng concentration. For a decreasing Ng concentration, AC8-Ct and AC8-Nt again exhibit the greatest relative change in CaM-binding. (F) Comparing the semi-isolated (dotted traces) to the competitive (solid traces) model shows that only in the competitive model does summed AC8 (AC8-Nt + AC8-Ct, dark red) mirror the loss in CaM-CaMKII binding as Ng concentration decreases.</p