Human defensins are cationic and Cys-rich antimicrobial proteins of 3-5 kDa expressed predominantly in neutrophils and epithelial cells. They broadly kill bacteria, fungi and certain enveloped viruses through disruption of the cytoplasmic membranes of invading microbes, thus playing important roles in phagocytosis and in protection of mucosal surfaces against microbial infections. An essential component in innate immunity, human defensins also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells, presumably through binding and activation of receptors for chemotaxis. All human defensins of known structure adopt a similar, three-stranded, anti-parallel 13-sheet conformation stabilized by three intra-molecular disulfide bridges. A distinct structural feature of these protein molecules is the spatial segregation of clusters of cationic residues from patches of hydrophobic side-chains in either monomeric or multimeric forms. The amphiphilic property, structurally maintained through disulfide bonding, is thought to be required for defensins to form multimeric ion-permeable pores in the cytoplasmic membranes of microorganisms, therefore functionally important. Due to important roles human defensins play in host immune response and the prospect of developing defensin-based, new classes of peptide antibiotics, it is prudent that we dissect the functional contributions of disulfide bonding in hBD3. Specific studies are envisaged as follows:
Specific Aim 1. Prepare all fifteen topological analogs of hBD3 with different disulfide connectivities. Various hBD3 polypeptides with reversible protection of selected Cys residues will be chemically synthesized, and oxidatively refolded into predefined topological structures.
Specific Aim 2. Test the hypothesis that antimicrobial activity of hBD3 is irrespective of whether or not, and how, disulfide bridges are paired. Antimicrobial assays will be performed to determine minimum inhibitory concentrations (MIC) using both Gram-positive and -negative bacteria.
Specific Aim 3. Test the hypothesis that chemotactic activity of hBD3 can be modulated by varying the topology of three disulfides. Chemotaxis indexes for these proteins will be evaluated, and the data will be correlated with binding affinities for known receptors.
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