The isolation, structure, and membrane interactions of biologically active peptides.

Abstract

The host-defence secretions of amphibians and the venoms of arachnids are an abundant source of biologically active peptides with a great potential for use in therapeutic pharmacology. Over millions of years of evolution, the chemical arsenals of a multitude of species have produced a vast collection of peptides that have potent and selective activities. The research presented in this thesis details the isolation, structure determination and mechanistic pathways of a selection of biologically active peptides. The southern brown tree frog Litoria ewingi occupies areas of the southeastern coast of Australia and Tasmania. Over a twelve month period, the peptide skin profile of a population of L. ewingii from Penola (South Australia) was determined using a combination of chromatography, tandem mass spectrometry and Edman degradation techniques. The peptide profiles of a L. ewingi from Penola show surprising differences relative to a population previously studied from the Adelaide hills, despite appearing to be morphologically identical. A total of six skin peptides were identified, four of which were unique; showing peptide sequence homology with peptides from Adelaide hills population. The evidence showed how a species can evolve separately after long periods of geographical isolation, how peptide profiling can be used to trace the migration of a species, and how new peptides can be discovered from different populations of a species. The antimicrobial meucin peptides were first identified using cDNA cloning of DNA from the venom gland of the ‘Lesser Asian scorpion’ Mesobuthus eupus mongolicus. These peptides exhibit cytolytic effects against a number of eukaryotic and prokaryotic cells at micromolar concentrations, and their peptide sequences share similarities with other antimicrobial peptides from scorpions, arthropods and amphibian species. The secondary structures of the meucin peptides were determined using 2-D NMR and molecular dynamics calculations. Both meucin peptides exhibit α-helical structure, and are amphipathic in nature. The study further shows how the length of the α-helical structure can as an antibiotic affect the cytolytic activity of the peptide, since meucin-18 is more potent than meucin-13. The C-terminal amide analogue of the peptide fallaxidin 4.l (fallaxidin 4.1a) isolated from the dermal secretions of Litoria fallax, is partially α-helical in nature, and shows potent activity against a wide range of yeast and bacteria (both Gram-positive and Gramnegative). This thesis uses solid-state NMR to detail the dynamic interactions of fallaxidin 4.1a with artificial lipid bilayers, and to explore the surface interactions of the peptides with eukaryotic (neutral) and prokaryotic (anionic) membranes. The solid state NMR and analysis using a quartz crystal microbalance indicated that the peptide acts via a surface interaction with neutral membranes and forms pores within anionic membranes at micromolar concentrations, indicating the specific pore forming mechanism by which the peptide interacts with anionic (prokaryotic) membranes. Rothein 1, an 11 residue neuropeptide from the dermal secretions of Litoria rothii, and two alanine substituted analogues, rothein 1.4 and 1.5; show differing activities via binding to CCK2 receptors. The structures of rothein 1.4 and 1.5 were determined using 2-D NMR and molecular dynamics calculations. Each peptide has a largely extended structure, with similarities to the structure of rothein 1. Two 10 residue, disulfidecontaining neuropeptides signiferin 1 and riparin 1 from dermal secretions of frogs of the Crinia genus, show potent smooth muscle and splenocyte activities. The dynamics of the interaction of signiferin 1, riparin1 and rothein 1 with artificial eukaryotic (neutral) lipid bilayer suspensions were probed using solid-state NMR, to emulate how a neuropeptide interacts with a cellular membrane surface prior to receptor binding. Solid-state NMR showed that rothein 1 had little effect on the mobility and orientation of the lipids, signiferin 1 interacted largely at the surface of the bilayers, and riparin 1 was partially inserted into the membrane. Rothein 1 is significantly less active than the disulphide peptides and more hydrophilic in nature; this is reflected in the interactions with bilayers. The disulphide peptides are more hydrophobic in character and the solid-state NMR indicated that they adhere to membranes.