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

除了G蛋白偶联受体亚型外，圆锥螺属的海洋蜗牛还能产生针对电压门控或配基门控离子通道的螺毒素，具有前所未有的选择性和特异性。本课题是对从圆锥体中分离得到的两种新型的含有g-羧谷氨酸(GLA)的芋螺毒素的结构和功能的研究。GLA是一种金属离子结合氨基酸，在哺乳动物维生素K依赖的凝血蛋白的膜结合特性中起着关键的结构作用。这种对合成GLA所需的酶机制的系统发育保存表明GLA在动物界中扮演着重要的角色。首先，电位法和荧光光谱学将被用来确定这些芋螺毒素所具有的金属离子结合常数和结合位点数。同时，利用圆二色谱和核磁共振氢谱对金属离子诱导的多肽主链和GLA残基侧链的结构扰动进行监测。这些芋螺毒素在钙诱导构象中的三维结构将用核磁共振波谱来确定。最后，该项目将研究二硫键支架和其他翻译后修饰的氨基酸赋予这些多肽的结构/功能意义，包括溴化色氨酸和糖基化苏氨酸。目前缺乏有关螺杆菌属内GLA和其他翻译后修饰氨基酸的结构/功能信息，这阻碍了神经学家、药理学家和结构生物学家了解芋螺毒素所固有的蛋白质受体识别的精细机制。在这个后基因组时代，对翻译后修饰的结构和功能含义的洞察变得越来越重要，因为科学家们试图弄清楚“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".