Calcium Binding Properties of Synthetic γ-Carboxyglutamic Acid-Containing Marine Cone Snail “Sleeper” Peptides, Conantokin-G and Conantokin-T^†

Total chemical synthesis of two Conus-derived peptides, conantokin-G (con-G), a 17-residue polypeptide containing five residues of γ-carboxyglutamic acid (Gla), and conantokin-T (con-T), a 21-residue polypeptide possessing four residues of Gla, was accomplished. Calcium binding isotherms were obtained for each peptide, and these differed considerably from each other. The binding isotherm for con-G was complex and could only be fit to degenerate models involving multiple Ca²⁺ binding sites. The data for Ca²⁺ binding to con-T was uniquely fit to a simple one-site model. In the case of con-G, circular dichroism (CD) studies revealed a polypeptide without observable α-helicity in the absence of Ca²⁺ and a dramatic shift to a high degree of α-helix at saturating Ca²⁺ concentrations. In contrast, apo-con-T possessed significant α-helical structure, and saturation with Ca²⁺ produced a less substantial change in its α-helical content. Titrations with Ca²⁺ of the change in α-helical content of con-T produced a C₅₀ value for Ca²⁺ that was essentially the same as its K_d from direct binding studies, demonstrating that occupancy of the single macroscopic binding site resulted in the conformational change. Similar titrations with con-G provided a C₅₀ value in concert with the K_d for binding of Ca²⁺ to this peptide. Moreover, in agreement with these particular Ca²⁺-induced structural changes, gel filtration analyses demonstrated significantly reduced hydrodynamic volumes of both of these polypeptides after saturation of their apo forms with Ca²⁺, with con-G showing a more pronounced change than con-T. One-dimensional ¹H-NMR spectra showed both line broadening and changes in chemical shifts of several peptide amide proton resonances after addition of Ca²⁺ to con-G, again suggestive of a large Ca²⁺-induced conformational change in this polypeptide. A derivative of con-G was synthesized with all amino acids present in the d-configuration (d-con-G). This variant peptide displayed Ca²⁺ binding isotherms nearly identical to those of con-G and underwent a Ca²⁺-induced conformational change very similar to that of con-G. Intracranial injections of con-G and con-T in young (<2 weeks) and older (3−4 weeks) mice produced the expected “sleep-like” and hyperactive effects, respectively. The variant, d-con-G, was inactive in these assays. These studies demonstrate that synthetic con-G and con-T possess their expected bioactivities and undergo large and defined conformational alterations in the presence of Ca²⁺. We propose that binding of Ca²⁺ to these polypeptides contributes to their ability to adopt a defined conformation, and this divalent cation-dependent conformation is necessary for their neuroactivities.