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.
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