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.

从多种圆锥（Magus，Striatus， 掩盖，图利帕，紫红色，辐射，加利福尼亚，纺织品， 地理，太平洋等）被识别，纯化和特​​征 在不同的项目中，将使用tertiobutyl-合成 氧气（BOC）或氟苯基甲基 - 氧核（FMOC）策略 固体支撑并使用离子交换纯化至高度纯度 和反相高压液相色谱。这些肽 将以许多分析技术（包括）来表征 正交色谱系统（例如，反相和离子交换）， 毛细管电泳（CZE），质谱（MS）和圆形 二分（CD）。我们将完成结构的特征 天然结合毒素肽通过比较其色谱行为 （Coelution实验）在不同条件下（包括CZE）， 它们的合成重复。在那些消性混合物的情况下 氨基酸的纳入合成重复，我们将 确定合成肽中氨基酸的手性 与天然酶毒素肽共同穿治。我们将确定何时 适用的，新型循环的二硫键桥接布置 使用A的合成肽（与天然肽相同） HPLC，化学序列分析，质谱和 部分还原技术。我们将比较不同的圆形 同一类蛋白毒素中合成肽的二分法光谱 帮助确定出色或支出结构以进一步 NMR或X射线晶体学研究（协作研究）。 最后，综合毒素肽的质谱（天然和合成） 纯化和特征在犹他大学（完整和完整 水解碎片）将在Salk Institute进行。这 我们提供的分析工具和程序包括多种多样 色谱程序，毛细管区电泳，合成 和化学水解策略的酶促耦合到质量 序列测定的光谱。这个核心，通过汇聚 建立组的合成和分析专业知识将启用 该计划项目的成员达到各自的目标 经济和及时的方式。优先顺序将决定 由计划项目总监和该核心的PI撰写的人会见面 经常。通过互联网的电子邮件链接被用作 需要。日常操作将由A. Gray Craig博士协调 犹他大学的Salk Institute和M McIntosh博士。