CORE--CHARACTERIZATION AND SYNTHESIS OF NOVEL CONOTOXINS

核心——新型芋螺毒素的表征与合成

• 批准号: 6610801
• 负责人: JEAN E RIVIER
• 金额: --
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610801
• 关键词: biomedical facility; capillary electrophoresis; circular dichroism; conotoxin; high performance liquid chromatography; mass spectrometry; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein folding; protein purification; protein structure function

Novel toxins and analogs from a number of species of Conus (magus, striatus, obscurus, tulipa, purpurascens, radiatus, textile, geographus, bocki and others) characterized or designed in the different projects will be synthesized using the Boc or Fmoc strategies. Folding conditions will be optimized to increase yields and facilitate purification. These peptides will be characterized with a number of analytical techniques including orthogonal chromatographic systems (e.g. reverse phase and ion exchange), capillary zone electrophoresis (CZE), chemical sequence analysis, mass spectrometry (MS) and circular dichroism (CD). We will complete the characterization of the structure of the natural conotoxin peptides by comparing their chromatographic behavior under different conditions (including CZE) with those of their synthetic replicates. 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 spectrometry and partial reduction techniques well established in our laboratories. For the purpose of identifying key residues in novel toxins, alanine scans and conventional substitutions will be carried out. Because secondary structures of most conotoxins are not always unique on an NMR time scale, attempts will be made to stabilize these structures using constrained N- and C-methylated amino acids and constrained scaffolds resulting from side chain to side chain bridging. We have found that retention or loss of activity as well as biological selectivity may be interpreted as resulting from favorable or unfavorable conformational constraints. Finally, mass spectrometry will be used extensively for the characterization of both native and synthetic conotoxins purified and characterized at the University of Utah and The Salk Institute. 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. The Program Project Director and the P! of this Core set priorities and communicate regularly. Day to day operations will be coordinated by Dr. W. Fischer at the Salk Institute and Dr. M. McIntosh at the University of Utah.

来自许多在不同项目中表征或设计的新型毒素和类似物（Magus，Striatus，Submustus，Tulipa，Tulipa，Tulipa，purpurascens，radiatus，textile，Geographus，Bocki等）将使用BOC或FMOC策略合成。折叠条件将被优化以提高产量并促进纯化。这些肽将以多种分析技术（包括正交色谱系统（例如反相和离子交换），毛细管区电泳（CZE），化学序列分析，质谱（MS）和圆形二色性（CD）（CD）来表征。我们将通过将不同条件（包括CZE）的色谱行为与它们的合成复制的形式行为进行比较，从而完成自然综合毒素肽的结构的表征。我们将 在适用的情况下，确定新型环状合成肽的二硫键桥接布置（与天然肽相同），使用HPLC，化学序列分析，质谱和部分还原技术的组合在我们的实验室中确定。为了鉴定新型毒素中的关键残基，将进行丙氨酸扫描和常规取代。由于大多数结构毒素的二级结构并不总是在NMR时间范围内独特，因此将尝试使用约束的N-和C-甲基化氨基酸和侧链到侧链桥梁引起的约束支架来稳定这些结构。我们发现保留或活动损失是 以及可以将生物选择性解释为由有利或不利的构象约束所产生的。 最后，质谱法将广泛用于在犹他大学和萨尔克学院纯化和表征的天然和合成结合毒素的表征。通过将既定小组的合成和分析专业知识汇总在一起，该核心将使该计划项目的成员能够在经济和及时实现各自的目标。 计划项目总监和P！此核心设定优先级并定期进行交流。日常操作将 由Salk Institute的W. Fischer博士和犹他大学的M. McIntosh博士协调。