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

芋螺属的海洋蜗牛产生芋螺毒素，其靶向电压门控或配体门控离子通道以及具有前所未有的选择性和特异性的G蛋白偶联受体亚型。本项目是对从纺织芋螺中分离的两种新的含g-羧基谷氨酸（Gla）的芋螺毒素的结构/功能进行研究。Gla是一种金属离子结合氨基酸，在定义哺乳动物维生素K依赖性凝血蛋白的膜结合特性中起关键的结构作用。这种合成Gla所必需的酶机制的遗传保存表明Gla在动物界中的重要作用。最初，电位法和荧光光谱法将被用来确定金属离子的结合常数和结合位点的数量所拥有的这些芋螺毒素。同时，将使用圆二色谱和1H NMR光谱监测金属离子诱导的肽骨架和Gla残基侧链的结构扰动。这些芋螺毒素在其钙诱导构象的三维结构将用NMR光谱法测定。最后，该项目将研究二硫键支架和其他后修饰氨基酸（包括溴化色氨酸和糖基化苏氨酸）赋予这些肽的结构/功能意义。目前缺乏有关芋螺属内Gla和其他后修饰氨基酸的结构/功能信息，这阻碍了神经科学家、药理学家和结构生物学家理解芋螺毒素固有的蛋白质-受体识别的精细机制。在这个后基因组时代，深入了解翻译后修饰的结构和功能意义变得越来越重要，因为科学家试图了解“功能是否遵循形式”。

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".