Solution NMR Studies Of Peptide Toxins From Cone Snails And Scorpion
Kumar, G Senthil
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Major constituents of the venom of various animals are peptidogenic in nature. Marine snails belonging to the species Conus are venomous predators that use small, structurally constrained peptides present in their venom for prey capture and defense. It is known that ~500 Conus species are present in nature and the venom of each of these Conus species is a complex mixture of nearly 100 peptides accounting for > 50,000 peptides with little overlap among the different species. The peptides isolated from the venom of Conus species are commonly known as conotoxins or conopeptides. Some of the common targets of these peptides include the different ion channels like Na+, K+, and Ca2+, and receptor subtypes such as nicotinic acetylcholine and NMDA receptors. The ion channels and receptor subtypes were targeted by conopeptides with high degree of specificity and selectivity. The structural information on the peptides from cone snails can prove to be a valuable starting tool for the understanding of the function of different ion channels and hence in the design of neuropharmacologically active drugs. Conotoxins are disulfide-rich peptides and the number of disulfide generally ranges from two to five. Based on the arrangement of cysteines in their primary sequence, they are classified into different superfamilies. The signal sequences of the precursors belonging to a particular superfamily are highly conserved and hence the members within the same family have, in common, the unique disulfide arrangement and pharmacological activity. Conotoxins are classified into eleven superfamilies till date. In order to understand the underlying the principles involved in the action of these peptides on different ion channels, one needs to know the three-dimensional structures which, in potential, will help in the identification of the pharmacophores responsible for the observed pharmacological activity. With the aim of studying the structure-activity relationships found among the conotoxins, we have initiated a study on the peptides isolated from the marine snails found in the Indian coastal waters. This thesis is focused in the structural studies of the peptide toxins from marine cone snails and a terrestrial scorpion. The tool used for the structural studies of these peptide toxins is Nuclear Magnetic Resonance Spectroscopy. Chapter 1 provides an overview of the peptide toxins found among various animal species with more emphasis on conotoxins and scorpion toxins. In addition, the rationale behind the present study has also been explained. Chapter 2 describes the structure determination of two conopeptides isolated from Conus amadis, δ-Am2766 and Am2735, which are active on mammalian sodium channels. The structural aspects and comparison with other known conopeptides belonging to the same superfamily as that of these two peptides have also been described. Solution NMR studies of Ar1446 and Ar1248, two conopeptides isolated from the species Conus araneosus have also been studied using Homonuclear NMR methods. Ar1446 is a three disulfide-bonded peptide. Our studies have revealed that this peptide has a novel disulfide connectivity not previously observed in the M superfamily or any other superfamily of conotoxins. The structural features of Ar1446 will be described along with the NMR studies on two-disulfide bonded peptide, Ar1248, belonging to the A-superfamily of conotoxins. The main problem faced in the kind of study of peptides isolated from natural sources is the amount that can be isolated and purified to homogeneity. In order to obtain large quantities of peptides, we have successfully used Cytochrome b5 as fusion host to clone, over express and purify these peptides using recombinant methods. The use of recombinant methods has aided in the preparation of isotopically enriched peptides. The use of cyt b5 as fusion host for the large scale production of some of the peptides from Indian marine snails is described in Chapter 4. A novel pharmacologically active linear peptide, Mo1659 isolated from Conus monile, have been studied using Heteronuclear NMR methods. This peptide was cloned, over expressed and purified using Cytochrome b5 as a fusion host. Another linear peptide, Mo1692 (also from Conus monile), has been prepared using the same method and was studied using Homonuclear NMR methods. Both these peptides were liberated from the fusion host using cyanogen bromide cleavage and were subsequently purified using RP-HPLC. The results of the biosynthetic preparation and NMR studies of these two peptides have been described in Chapter 5. Chapter 6 describes the solution structure determination of a novel scorpion toxin characterized in the venom of the Indian red scorpion Buthus tamulus. The cloning, over expression, folding and purification of BTK-2 is described here. The structure and the function of this recombinantly produced BTK-2 will also be described.