Neuronal nicotinic acetylcholine receptors (nAChRs) are of crucial physiological importance and they have also been implicated in a number of pathological conditions thus representing important targets for pharmacological intervention. Knowledge of the structure-function relationships of receptor: ligand interactions can facilitate design of improved pharmacological tools and drugs. The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. The toxins are unusually rich in post-translationally modified amino acids, the significance of which is not well understood. The aims of this thesis were to discover new conotoxin antagonists of nAChRs, to investigate the roles of post-translationally modified amino acids in α-conotoxins, and to improve prediction of sites of posttranslational modifications in conotoxins. The discovery and characterisation of several novel α-conotoxins and posttranslationally modified peptides identified by a functional assay and by LC/MS of crude venom extracts from several species of cone snails was described (Chapter 2). The most interesting species was Conus anemone with activities at a range of neuronal nAChR subtypes. Three sulfated α-conotoxins-AnIA, AnIB and AnIC- were identified by LC/MS and assay directed fractionation, and sequenced after purification. Synthetic AnIB exhibited subnanomolar potency at the rat α3β2 nAChR and was 200-fold less active on the rat α7 nAChR. Reassessment of Conus aulicus crude venom revealed a sulfotyrosinylated variant of AuIA. Prediction of the likelihood of sulfation of tyrosine residues in α-conotoxins was consistent with observations of sulfotyrosinylation. The roles of some post-translationally modified amino acid residues, including sulfotyrosine, y-carboxyglutamic acid and hydroxyproline, were investigated by chemical and functional characterisation of variants of known α-conotoxins (Chapter 3). AnIB was selected as an example of a sulfotyrosinylated α-conotoxin, and activities of sulfated and non-sulfated AnIB variants at two murine nAChR subtype combinations were compared. The unsulfated peptide [Tyr16]AnIB showed a two-fold and ten-fold decrease in activity at α3β2 (IC50 0.6 nM) and α7 (IC50 836 nM) nAChRs, respectively. GID was selected as a model for y-carboxyglutamic acid and hydroxyproline in α-conotoxins. Substitution of glutamic aid or glycine for y- carboxyglutamic acid at position 4 of GID, and substitution of proline for hydroxyproline at position 16 of GID, did not markedly alter affinity for nAChRs. Several well-characterised α-conotoxins were selected to study the effects of substitution of hydroxyproline for the conserved proline in Loop I, and this change in variants of [A10L]PnIA, PnIA and EI, but not ImI, resulted in loss of activity. The post-translationally modified residue provided a useful tool to further probe the interactions between α-conotoxins and nAChRs or their homologue, acetylcholine binding protein (AChBP). The discovery, and the chemical and functional characterisation of three novel conotoxins - αD-VxXIIA, αD-VxXIIB and αD-VxXIIC - purified from the venom of Conus vexillum was described (Chapter 4). The αD-conotoxin family is a new category of peptides in the venom of some Conus species from Clade XII. Each toxin was observed as an ~11 kDa protein by LC/MS, size exclusion chromatography and SDS-PAGE. The peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudohomodimers of paired 47– 50 residue peptides. The toxin subunits exhibited a novel arrangement of ten conserved cystine residues and post-translational modifications (y-carboxyglutamic acid and hydroxyproline) contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that αD-VxXIIA, αD-VxXIIB and αD-VxXIIC are potent inhibitors of nAChRs, with selectivity for α7 and β2- containing neuronal nAChR subtypes. These dimeric conopeptides appear to be a fifth and highly divergent structural class of conotoxins targeting nAChRs. Several cDNA sequences encoding αD-conotoxins in the venom of four species, C. vexillum, Conus capitaneus, Conus miles and Conus mustelinus, were determined (Chapter 5). The complete nucleic acid coding sequence for VxXIIA from C. vexillum was obtained. Related prepropeptide sequences, obtained by PCR and RACE analyses, shared the expected features of a moderately conserved signal peptide region and a variable mature toxin region. The signal peptide sequences displayed more heterogeneity than previously reported for other conotoxin superfamilies, with differing signal peptide sequence motifs defining two distinct groups of αD-conotoxin precursors. The predicted mature toxin sequences could be matched with partial Nterminal peptide sequences of components with nAChR antagonist activity, allowing resolution of the previously incomplete study. The discovery and characterisation of nAChR antagonists described in this study has contributed new tools to further study the structurefunction relationships of the ligandreceptor interactions, and the roles of post-translationally modified residues in conotoxins. The findings have suggested that naturally occurring post-translationally modified residues in α-conotoxins are not always critical to function, although hydroxyproline substitution at the position of the conserved proline in Loop I of most α-conotoxins is typically not tolerated. The single sites of hydroxyproline and y- carboxyglutamic acid in some α-conotoxins are in contrast to variable modification of proline and glutamic acid at multiple positions in many larger conotoxins. The newly described αD-conotoxins, together with their precursor sequences, represent a useful model for the further study of the roles of these post-translational modifications in conotoxins.