This research will generate detailed information on the solution structures of a recently discovered family of antimicrobial peptides known as defensins. These peptides have been isolated from phagocytic leukocytes of rabbits, guinea pigs and humans and form part of the oxygen-independent mammalian defense system. The ten peptides that have been characterized posses potent activities against a broad spectrum of microbes including bacteria, fungi, and enveloped viruses. These homologous peptides are all cysteine- and arginine-rich, 29-34 residues in length and have 8 conserved residues in the whole family. The defensins vary greatly in their potency and their range of activities. Their functional diversity most likely arises from structural variations between individual molecules. This project will probe the structure and dynamics of the defensins in solution with goal of searching for correlations between variations in structure, and distinct biological function. The primary technique that will be used to determine the structure of defensins is nuclear magnetic resonance (NMR) spectroscopy. Two-dimensional (2D) nuclear Overhauser effect experiments will be used to obtain proton-proton internuclear distances less than 4.5 angstroms. Additional 2D NMR experiments will measure spin-spin coupling constants which give information on dihedral angles in the molecule. This dihedral angle and distance information then serves as the input for distance geometry calculations which generate three-dimensional structures of the molecules in solution. This family of peptides also provides an ideal natural laboratory for studying the effects of substitution of individual amino acids on the folding, structure, and dynamics of peptides in solution. The solution structures of the defensin family will provide a unique opportunity for probing the molecular properties that give rise to the biological function of these peptides and may help elucidate their mechanism of action in vivo.
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