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若干种圆锥动物的新型毒素和类似物(圆锥虫、条纹圆锥虫、暗圆锥圆锥虫、郁金香、紫腹圆锥虫、放射状圆锥虫、纺织、地理圆锥虫和其他)。折叠条件将进行优化，以提高产量和促进纯化。这些多肽将用多种分析技术进行表征，包括正交色谱系统(如反相和离子交换)、毛细管区带电泳法(CZE)、化学序列分析、质谱学(MS)和圆二色谱(CD)。我们将通过比较天然芋螺毒素多肽在不同条件下的层析行为(包括CZE)和它们的合成复制品来完成对其结构的表征。我们会 在适用的情况下，使用高效液相色谱、化学序列分析、质谱分析和我们实验室建立的部分还原技术相结合的方法来确定新型环合合成肽(经证明与天然多肽相同)的二硫键桥联排列。为了识别新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核磁共振时间尺度上并不总是唯一的，因此将尝试使用约束的N-和C-甲基化氨基酸和从侧链到侧链桥联的约束支架来稳定这些结构。我们发现，活动的保留或丧失 生物选择性可被解释为有利或不利构象限制的结果。最后，质谱学将广泛用于鉴定在犹他大学和索尔克研究所提纯和鉴定的天然和合成芋螺毒素。这一核心，通过将现有小组的综合和分析专业知识结合在一起，将使该计划项目的成员能够在经济上和及时地实现各自的目标。计划项目总监和P！确定优先事项，并定期进行沟通。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。

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.