CORE--CHARACTERIZATION AND SYNTHESIS OF NOVEL CONOTOXIN PEPTIDES

核心——新型芋螺毒素肽的表征与合成

• 批准号: 6564577
• 负责人: JEAN E RIVIER
• 金额: $ 14.53万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564577
• 关键词: Gastropoda; animal poison; biomedical facility; calcium channel blockers; capillary electrophoresis; circular dichroism; conotoxin; high performance liquid chromatography; ion exchange chromatography; mass spectrometry; neurotoxins; peptide chemical synthesis; peptide structure; protein purification; protein sequence; synthetic peptide; zonal electrophoresis

Selected toxins from a number of species of Conus (magus, striatus, obscuras, tulipa, purpurascens, radiatus, californicus, textile, geographus, pacificus and others) identified, purified and characterized in the different projects will be synthesized using the tertiobutyl- oxycarbonyl (Boc) or fluorenylmethyl-oxicarbonyl (Fmoc) strategies on solid supports and purified to high degree of purity using ion exchange and reverse phase high pressure liquid chromatographies. These peptides will be characterized with a number of analytic techniques including orthogonal chromatographic systems (e.g. reverse phase and ion exchange), capillary zone electrophoresis (CZE), mass spectroscopy (MS) and circular dichroism (CD). We will complete the characterization of the structure of the natural conotoxin peptides by comparing their chromatographic behavior (coelution experiments) under different conditions (including CZE) with those of their synthetic replicates. In those cases where racemic mixtures of amino acids are incorporated into synthetic replicates, we will determine the chirality of the amino acids in the synthetic peptide which co-elutes with the native conotoxin peptide. We will determine, when applicable, the disulfide bridging arrangement of the novel cyclic synthetic peptides (shown to be identical to the native peptides) using a combination of HPLC, chemical sequence analysis, mass spectroscopy and partial reduction techniques. We will compare the different circular dichroism spectra of synthetic peptides in the same class of conotoxin to help identify outstanding or outlaying structures for further investigation by NMR or X-ray crystallography (collaborative studies). Finally, mass spectroscopy of conotoxin peptides (native and synthetic) purified and characterized at the University of Utah (both intact and hydrolyzed fragments) will be carried out at the Salk Institute. The analytical tools and procedures which we make available include a variety of chromatographic procedures, capillary zone electrophoresis, synthesis and enzymatic of chemical hydrolysis strategies coupled to mass spectroscopy for sequence determination. This core, by bringing together the synthetic and analytical expertise of an established group will enable members of this program project to reach their respective goals both economically and in a timely fashion. Order of priorities will be decided by the Program Project Director and the PI of this core who will meet regularly. An electronic mail link through internet is being used as needed. Day to day operations will be coordinated by Dr. A. Grey Craig at the Salk Institute and Dr. M McIntosh at the University of Utah.

从芋螺（magus，striatus， obscuras，郁金香，purpurascens，辐射，加州，纺织品， geographus，pacificus等）进行鉴定、纯化和表征 在不同的项目中，将使用叔丁基- 氧羰基（Boc）或芴基甲基-氧羰基（Fmoc）策略 固体载体，并使用离子交换纯化至高纯度 和反相高压液相色谱法。这些肽 将采用多种分析技术，包括 正交色谱系统（例如反相和离子交换）， 毛细管区带电泳（CZE）、质谱（MS）和循环质谱（CRE） 二向色性（CD）。我们将完成的结构表征 通过比较天然芋螺毒素肽的色谱行为， （共洗脱实验）在不同条件下（包括CZE）， 他们的合成复制品。在那些外消旋混合物 的氨基酸被纳入合成复制品，我们将 确定合成肽中氨基酸的手性， 与天然芋螺毒素肽共洗脱。我们将决定，当 适用的，新的环状化合物的二硫键桥连排列 合成肽（显示与天然肽相同）， HPLC、化学序列分析、质谱法和 部分还原技术我们将比较不同的循环 在相同种类的芋螺毒素中合成肽的二色性光谱， 帮助确定未完成或支出结构， 通过NMR或X射线晶体学进行研究（合作研究）。 最后，芋螺毒素肽（天然和合成）的质谱 纯化并在犹他州大学表征（完整和 水解片段）将在索尔克研究所进行。的 我们提供的分析工具和程序包括各种 色谱程序，毛细管区带电泳，合成 和酶的化学水解策略耦合到质量 用于序列测定的光谱学。这个核心，通过汇集 一个已建立小组的综合和分析专门知识将使 该项目的成员，以达到各自的目标， 经济上和及时地。优先顺序将由 由计划项目总监和该核心的PI进行，他们将与 定期。通过互联网的电子邮件链接正在被用作 needed.日常工作由A博士负责协调。格雷克雷格在 索尔克研究所和犹他州大学的麦金托什博士。