Isothermal Calorimetry (ITC) analysis indicates a 2∶1 stoichiometry of NSCaTE:CaM in the absence of IQ, and a 1∶1 NSCaTE:CaM stoichiometry when CaM is first pre-bound to IQ.

<p>(A) Representative raw sample data for several CaM-peptide titrations. (B) Summary Table of ITC data. NSCaTE or IQ peptides were titrated into Ca²⁺-CaM alone or Ca²⁺-CaM pre-bound to a competing peptide at a 1∶1 ratio. All binding reactions exhibited negative enthalpy under the experimental conditions used (exothermic, ΔH <0). Despite the lack of mobility shift of Ca²⁺-CaM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061765#pone-0061765-g007" target="_blank">Figure 7</a>), NSCaTE does bind Ca²⁺-CaM alone. LCa_v1 and Ca_v1.2 IQ peptides have a higher affinity (Kd = 80 nM and 130 nM, respectively) for Ca²⁺-CaM than do either of the NSCaTE peptides (Kd = 0.83 µM and 3.24 µM for Ca_v1.2 and LCa_v1 NSCaTEs). Both mammalian NSCaTE_Cav1.2 and snail NSCaTE_LCav1 is able to bind to Ca²⁺-CaM pre-bound to IQ motifs, and both IQ peptides are able to bind to CaM when it is first bound to NSCaTE, which is consistent with the possibility of a NSCaTE-CaM-IQ complex <i>in vivo</i>. N values indicate that NSCaTE_LCav1 and NSCaTE_Cav1.2 can bind to Ca²⁺-CaM in a 2∶1 stoichiometry, consistent with NSCaTE possibly binding simultaneously to both the N- and C-lobes of CaM, as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061765#pone.0061765-Liu1" target="_blank">[30]</a>.