Structure/Function Investigation of Gamma-Carboxyglutamic Acid-Containing Conotoxins

Marine Biological Laboratory, Woods Hole MA

Investigators

Abstract

Marine snails of the genus Conus produce conotoxins that target voltage- or ligand-gated ion channels in addition to G-protein coupled receptor subtypes with unprecedented selectivity and specificity. This project is a structure/function investigation of two novel g-carboxyglutamic acid (Gla)-containing conotoxins, isolated from Conus textile. Gla is a metal-ion binding amino acid that plays a critical structural role in defining the membrane binding properties of mammalian vitamin K-dependent blood coagulation proteins. This phylogenic preservation of the enzymatic machinery necessary to synthesize Gla suggests an important role for Gla within the animal kingdom. Initially, potentiometry and fluorescence spectroscopy will be used to determine the metal-ion binding constants and number of binding sites possessed by these conotoxins. Simultaneously, metal-ion induced structural perturbations of the peptide backbone and Gla residue side chain will be monitored using circular dichroism and 1H NMR spectroscopy. The three-dimensional structure of these conotoxins in their calcium-induced conformation will be determined with NMR spectroscopy. Finally, the project will examine the structure/function significance conferred upon these peptides by the disulfide bond scaffold and other post-translationally modified amino acids including; brominated tryptophan and glycosylated threonine. The current paucity of structure/function information pertaining to Gla and other post-translationally modified amino acids within the genus Conus has prevented neuroscientists, pharmacologists and structural biologists from understanding the refined mechanism of protein-receptor recognition that is innate to conotoxins. Insight into the structural and functional implications of post-translational modifications has become increasingly important in this post-genomic era, as scientists attempt to understand if "function follows form".