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.

从圆锥虫的一些种类中精选出的毒素(魔芋，纹状体， 暗色、郁金香、紫罗兰、放射状、杯状、纺织类、 地理、太平洋等)鉴定、提纯和表征 在不同的项目中将使用叔丁基- 氧羰基(Boc)或荧甲氧羰基(Fmoc)策略 固体载体，使用离子交换提纯至高纯度 和反相高压液相色谱仪。这些多肽 将以许多分析技术为特征，包括 正交色谱系统(如反相和离子交换)， 毛细管区带电泳(CZE)、质谱学(MS)和环状图谱 二向色性(CD)。我们将完成对结构的表征 比较天然芋螺毒素多肽的层析行为 (洗脱实验)在不同条件下(包括CZE) 它们的合成复制体。在外消旋混合物的情况下 的氨基酸被加入到合成复制品中，我们将 测定合成肽中氨基酸的手性 与天然芋螺毒素多肽共洗脱。我们将确定，何时 适用的，新型环状化合物的二硫键桥联排列 合成多肽(显示与天然多肽相同)使用 高效液相色谱、化学序列分析、质谱学和电子计算机联用 部分还原技术。我们将比较不同的循环 同一类芋螺毒素合成肽的二色性光谱 帮助确定未完成或未完成的结构，以便进一步 通过核磁共振或X射线结晶学(合作研究)进行研究。 芋螺毒素多肽(天然和人工合成)的质谱学研究 在犹他州大学提纯和鉴定(完好无损和 将在索尔克研究所进行。这个 我们提供的分析工具和程序包括各种 层析程序，毛细管区带电泳法，合成 和酶的化学水解策略相结合的质量 用于序列测定的光谱学。这一核心，通过将 现有小组的综合和分析专业知识将使 该计划的成员计划实现他们各自的目标 经济上和及时的。将决定优先顺序 由计划项目总监和此核心的PI会面 定期的。通过互联网的电子邮件链接被用作 需要的。日常运作将由A.Grey Craig博士在 索尔克研究所和犹他大学的M·麦金托什博士。