Structural studies on the psi-conotoxins and the secondary metabolites of a marine tunicate

Each project described in this dissertation explores the three-dimensional structure of the psi-conotoxins, the structural and neurotoxicological impact of each loop region of the psi-conotoxins, or the chemical structure of metabolites isolated from a marine ascidian. One of the characteristic features of the conotoxins is a high density of disulfide bridges in each peptide. The conformational properties of the disulfide bridges in previously determined conotoxin structures are reviewed in the first chapter. The second chapter describes the use of selective 13C-labeling of cysteine residues as a means of resolving resonance overlap problems in NMR for conotoxins through the use of 13C-filtering and 13C-editing. This chapter also describes the determination of a revised structure for psi-conotoxin PIIIE. Interproton distance restraints were derived from NOESY correlations through relaxation matrix calculations. A new set of structures were produced that were more precise, less strained, and better correlated with the experimental data. The revised structures differed from the original structures in the distribution of positive charges. The third chapter describes the structure determination of psi-conotoxin PIIIF using the same methodologies applied to psi-PIIIE. The structure of psi-PIIIF is very similar to that of psi-PIIIE. The two toxins differ in the degree of convergence of the chain termini, the distribution of polarity on the peptide surfaces, and in the degree of conformational freedom of the disulfide bridges. The fourth and fifth chapters explore the structure and activity of a series of chimeric mutant psi-conotoxins incorporating loop regions of both psi-PIIE and psi-PIIIF. The second loop region of the psi-conotoxins is the prime determinant of the degree of conformational freedom in the disulfide bridges. The binding of the psi-conotoxins and chimeric mutants to nAChR from the electric organ of Torpedo californica expressed on Xenopus oocytes was explored through electrophysiology. The third loop region had the largest impact on inhibitory potency. The first loop also affected potency. The sixth chapter describes studies exploring the secondary metabolites produced by a previously undescribed Eudistoma> species of ascidian. A series of guanidinium-containing beta-carbolines were discovered. Other metabolites isolated include a polyhydroxylated ?-carboline, a brominated tyramine, and a methylated isoxanthopterin.