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 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 alpha-helicity in the absence of Ca2+ and a dramatic shift to a high degree of alpha-helix at saturating Ca2+ concentrations. In contrast, apo-con-T possessed significant alpha-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 C50 value for Ca2+ that was essentially the same as its Kd 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 Kd for binding of Ca2+ to this peptide. Moreover, in agreement with these particular Ca(2+)-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-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 Ca(2+)-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 Ca(2+)-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.
Prorok
M
M
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Warder
S E
SE
Blandl
T
T
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D020916:Conotoxins; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012908:Snails / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism
D020916:Conotoxins; D036341:Intercellular Signaling Peptides and Proteins; D008978:Mollusk Venoms; D010455:Peptides; D010456:Peptides, Cyclic; C063571:conantokin-T; D015055:1-Carboxyglutamic Acid; C055407:conotoxin GV; D002118:Calcium