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-car boxyglutamic acid (Gla), and conantokin-T (con-T), a 21-residue polype ptide possessing four residues of cia, was accomplished. Calcium bindi ng isotherms were obtained for each peptide, and these differed consid erably from each other, The binding isotherm for con-G was complex and could only be fit to degenerate models involving multiple Ca2+ bindin g sites, The data for Ca2+ binding to con-T was uniquely fit to a simp le one-site model, In the case of con-G, circular dichroism (CD) studi es revealed a polypeptide without observable alpha-helicity in the abs ence of Ca2+ and a dramatic shift to a high degree of alpha-helix at s aturating Ca2+ concentrations. Tn contrast, apo-con-T possessed signif icant a-helical structure, and saturation with Ca2+ produced a less su bstantial change in its alpha-helical content. Titrations with Ca2+ of the change in alpha-helical content of con-T produced a C-50 value fo r Ca2+ that was essentially the same as its K-d from direct binding st udies, demonstrating that occupancy of the single macroscopic binding site resulted in the conformational change, Similar titrations with co n-G provided a C-50 value in concert with the K-d for binding of Ca2to this peptide, Moreover, in agreement with these particular Ca2+-ind uced structural changes, gel filtration analyses demonstrated signific antly reduced hydrodynamic volumes of both of these polypeptides after saturation of their apo forms with Ca2+, with con-G showing a more pr onounced change than con-T, One-dimensional H-1-NMR spectra showed bot h line broadening and changes in chemical shifts of several peptide am ide proton resonances after addition of Ca2+ to con-G, again suggestiv e of a large Ca2+-induced conformational change in this polypeptide. A derivative of con-G was synthesized with all amino acids present in t he D-configuration (D-con-G). This variant peptide displayed Ca2+ bind ing isotherms nearly identical to those of con-G and underwent a Ca2+- induced conformational change very similar to that of con-G. Intracran ial injections of con-G and con-T in young (<2 weeks) and older (3-4 w eeks) mice produced the expected ''sleep-like'' and hyperactive effect s, respectively, The variant, D-con-G, was inactive in these assays. T hese studies demonstrate that synthetic con-G and con-T possess their expected bioactivities and undergo large and defined conformational al terations in the presence of Ca2+, We propose that binding of Ca2+ to these polypeptides contributes to their ability to adopt a defined con formation, and this divalent cation-dependent conformation is necessar y for their neuroactivities.