Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic alpha-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly alpha-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide-membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA 2 ) and cytosolic phospholipase A 2 (cPLA 2 ) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA 2 and cPLA 2 align or are similar to ExoU sequences. Sitespeci®c mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid pro®les and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modi®cation by eukaryotic factors.

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The ferric enterobactin receptor (FepA) is a high-affinity ligand-specific transport protein in the outer membrane of Gram-negative bacteria. Deletion of the cell-surface ligand-binding peptides of FepA generated mutant proteins that were incapable of high-affinity uptake but that instead formed nonspecific, passive channels in the outer membrane. Unlike native FepA, these pores acted independently of the accessory protein TonB, which suggests that FepA is a gated porin and that TonB acts as its gatekeeper by facilitating the entry of ligands into the FepA channel. The sequence homology among TonB-dependent proteins suggests that all ligand-specific outer membrane receptors may function by this gated-porin mechanism.

Myelin basic protein is a candidate autoantigen in multiple sclerosis. One of its dominant antigenic epitopes is segment Pro 85 to Pro 96 (human sequence numbering, corresponding to Pro 82 to Pro 93 in the mouse). There have been several, contradictory predictions of secondary structure in this region; either ␤-sheet, ␣-helix, random coil, or combinations thereof have all been proposed. In this paper, molecular dynamics and site-directed spin labeling in aqueous solution indicate that this segment forms a transient ␣-helix, which is stabilized in 30% trifluoroethanol. When bound to a myelin-like membrane surface, this antigenic segment exhibits a depth profile that is characteristic of an amphipathic ␣-helix, penetrating up to 12 Å into the bilayer. The ␣-helix is tilted ϳ9°, and the central lysine is in an ideal snorkeling position for side-chain interaction with the negatively charged phospholipid head groups.Multiple sclerosis (MS) 1 is thought to be an autoimmune disease characterized by chronic inflammatory response against myelin in the central nervous system. There is significant evidence that myelin basic protein (MBP) is a candidate antigen for T-cells and autoantibodies in MS (1). The 18.5-kDa isoform of MBP maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers (2, 3). The mechanism and sites that are important for membrane adhesion are not known.The level of anti-MBP antibodies is increased in the cerebrospinal fluid of patients with active MS (4), as well as in 96% of patients with relapsing and chronic MS (5). An MBP region between Pro 85 and Pro 96 (human sequence numbering) was identified to be a minimal B cell epitope and a T cell epitope for HLA DR2b (DRB1*1501)-restricted T cells that recognize the protein (1, 6). This epitope overlaps with the DR2a-restricted epitope for T-cells reactive to MBP residues 87-106 (7). Experimental treatments for MS based on peptide mimetics of MBP have focused on this region of the protein (8).In solution, MBP is "intrinsically unstructured" (or "natively unfolded") (9). Upon binding to detergents or lipids, the levels of ␤-sheet and especially ␣-helical structure increase dramatically (10, 11). Presently the tertiary structure of MBP is unknown, with the most detailed predictions coming from Martenson (12) and Stoner (13) in the mid-1980s. In these models, as well as in a newer model based upon further research on an electron microscopy single-particle reconstruction, residues 86 -92 are found in a ␤-sheet (14, 15). Using [ 1 H]NMR and circular dichroic spectropolarimetry of various MBP peptides with detergent micelles, Mendz et al. (16) suggested that there were discrete interaction sites in the protein, one of which could be a helix between residues 87 and 97. Based on the arrangement of hydrophobic and hydrophilic residues, Warren et al. (6) predicted that this epitope of MBP was an amphipathic ␣-helix located at the interface between the oligodendrocyte cytoplasm and the me...