CORE--CHARACTERIZATION AND SYNTHESIS OF NOVEL CONOTOXINS

将使用Boc或Fmoc策略合成在不同项目中表征或设计的来自许多芋螺物种（magus、striatus、obscurus、tulipa、purpurascens、radiatus、textile、geographus、bocki等）的新型毒素和类似物。将优化折叠条件以增加产率并促进纯化。这些肽将采用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）下的色谱行为与合成复制品的色谱行为，完成天然芋螺毒素肽结构的表征。我们将 在适用时，使用我们实验室已建立的HPLC、化学序列分析、质谱和部分还原技术的组合，确定新型环状合成肽（与天然肽相同）的二硫键桥连排列。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。由于大多数芋螺毒素的二级结构在NMR时间尺度上并不总是独特的，因此将尝试使用受约束的N-和C-甲基化氨基酸和由侧链到侧链桥接产生的受约束的支架来稳定这些结构。我们发现，保持或丧失活性， 生物选择性也可以解释为有利或不利的构象限制。 最后，质谱法将被广泛用于表征天然和合成芋螺毒素纯化和表征在犹他州大学和索尔克研究所。这个核心，通过汇集一个既定的小组的合成和分析专业知识，将使该计划项目的成员能够经济和及时地实现各自的目标。 项目经理和P！本核心设定优先事项并定期沟通。日常运营将 由W博士协调。索尔克研究所的费舍尔和M.麦金托什在犹他州大学。

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.