Calcium binding properties of synthetic gamma-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 gamma-carboxyglutamic acid (Gla), and conantokin-T (con-T), a 21-residue polypeptide possessing four residues of cia, 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 alpha-helicity in the absence of Ca2+ and a dramatic shift to a high degree of alpha-helix at saturating Ca2+ concentrations. Tn contrast, apo-con-T possessed significant a-helical structure, and saturation with Ca2+ produced a less substantial change in its alpha-helical content. Titrations with Ca2+ of the change in alpha-helical content of con-T produced a C-50 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 C-50 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 H-1-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.