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.

在不同项目中表征或设计的来自许多圆锥芋属物种（magus、striatus、obscurus、tulipa、purpurascens、radiatus、textile、geographus、bocki 等）的新型毒素和类似物将使用 Boc 或 Fmoc 策略进行合成。将优化折叠条件以提高产量并促进纯化。这些肽将通过多种分析技术进行表征，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）、化学序列分析、质谱（MS）和圆二色性（CD）。我们将通过比较天然芋螺毒素肽在不同条件（包括 CZE）下的色谱行为与其合成重复品的色谱行为，完成对天然芋螺毒素肽结构的表征。我们将 在适用时，使用我们实验室成熟的 HPLC、化学序列分析、质谱和部分还原技术的组合来确定新型环状合成肽（显示与天然肽相同）的二硫键桥接排列。为了鉴定新型毒素中的关键残留物，将进行丙氨酸扫描和常规替代。由于大多数芋螺毒素的二级结构在 NMR 时间尺度上并不总是唯一的，因此将尝试使用受限的 N- 和 C- 甲基化氨基酸以及由侧链与侧链桥接产生的受限支架来稳定这些结构。我们发现，活性的保留或丧失 生物选择性可以解释为有利或不利的构象限制的结果。 最后，质谱法将广泛用于犹他大学和索尔克研究所纯化和表征的天然和合成芋螺毒素的表征。该核心通过汇集现有团队的综合和分析专业知识，将使该计划项目的成员能够经济且及时地实现各自的目标。 项目项目总监和 P!该核心成员设定优先事项并定期沟通。日常运营将 由索尔克研究所的 W. Fischer 博士和犹他大学的 M. McIntosh 博士协调。