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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI027026-02
Application #
3141069
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1988-03-01
Project End
1993-02-28
Budget Start
1989-03-01
Budget End
1990-02-28
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Zimmermann, G R; Legault, P; Selsted, M E et al. (1995) Solution structure of bovine neutrophil beta-defensin-12: the peptide fold of the beta-defensins is identical to that of the classical defensins. Biochemistry 34:13663-71
Skalicky, J J; Selsted, M E; Pardi, A (1994) Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics. Proteins 20:52-67
Skalicky, J J; Metzler, W J; Ciesla, D J et al. (1993) Solution structure of the calcium channel antagonist omega-conotoxin GVIA. Protein Sci 2:1591-603
Pardi, A; Zhang, X L; Selsted, M E et al. (1992) NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1. Biochemistry 31:11357-64
Zhang, X L; Selsted, M E; Pardi, A (1992) NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1. Biochemistry 31:11348-56
Heus, H A; Pardi, A (1991) Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science 253:191-4
Huang, L H; Cheng, H; Pardi, A et al. (1991) Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX. Biochemistry 30:7402-9
Kominos, D; Bassolino, D A; Levy, R M et al. (1990) Analysis of side-chain conformational distributions in neutrophil peptide-5 NMR structures. Biopolymers 29:1807-22
Pardi, A; Galdes, A; Florance, J et al. (1989) Solution structures of alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy. Biochemistry 28:5494-501
Levy, R M; Bassolino, D A; Kitchen, D B et al. (1989) Solution structures of proteins from NMR data and modeling: alternative folds for neutrophil peptide 5. Biochemistry 28:9361-72

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