CORE--CHARACTERIZATION AND SYNTHESIS OF NOVEL CONOTOXIN PEPTIDES

点击翻译按钮获取中文摘要

Selected toxins from a number of species of Conus (magus, striatus, obscuras, tulipa, purpurascens, radiatus, californicus, textile, geographus, pacificus and others) identified, purified and characterized in the different projects will be synthesized using the tertiobutyl- oxycarbonyl (Boc) or fluorenylmethyl-oxicarbonyl (Fmoc) strategies on solid supports and purified to high degree of purity using ion exchange and reverse phase high pressure liquid chromatographies. These peptides will be characterized with a number of analytic techniques including orthogonal chromatographic systems (e.g. reverse phase and ion exchange), capillary zone electrophoresis (CZE), mass spectroscopy (MS) and circular dichroism (CD). We will complete the characterization of the structure of the natural conotoxin peptides by comparing their chromatographic behavior (coelution experiments) under different conditions (including CZE) with those of their synthetic replicates. In those cases where racemic mixtures of amino acids are incorporated into synthetic replicates, we will determine the chirality of the amino acids in the synthetic peptide which co-elutes with the native conotoxin peptide. 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 spectroscopy and partial reduction techniques. We will compare the different circular dichroism spectra of synthetic peptides in the same class of conotoxin to help identify outstanding or outlaying structures for further investigation by NMR or X-ray crystallography (collaborative studies). Finally, mass spectroscopy of conotoxin peptides (native and synthetic) purified and characterized at the University of Utah (both intact and hydrolyzed fragments) will be carried out at the Salk Institute. The analytical tools and procedures which we make available include a variety of chromatographic procedures, capillary zone electrophoresis, synthesis and enzymatic of chemical hydrolysis strategies coupled to mass spectroscopy for sequence determination. 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. Order of priorities will be decided by the Program Project Director and the PI of this core who will meet regularly. An electronic mail link through internet is being used as needed. Day to day operations will be coordinated by Dr. A. Grey Craig at the Salk Institute and Dr. M McIntosh at the University of Utah.