The LAH4 family of histidine-rich peptides exhibits potent antimicrobial and DNA transfection activities, both of which require interactions with cellular membranes. The bilayer association of the peptides has been shown to be strongly pH-dependent, with in-planar alignments under acidic conditions and transmembrane orientations when the histidines are discharged. Therefore, we investigated the pH- and temperature-dependent conformations of LAH4 in DPC micellar solutions and in a TFE/PBS solvent mixture. In the presence of detergent and at pH 4.1, LAH4 adopts helical conformations between residues 9 and 24 concomitantly with a high hydrophobic moment. At pH 6.1, a helix-loop-helix structure forms with a hinge encompassing residues His¹⁰-Ala¹³. The data suggest that the high density of histidine residues and the resulting electrostatic repulsion lead to both a decrease in the pK values of the histidines and a less stable α-helical conformation of this region. The hinged structure at pH 6.1 facilitates membrane anchoring and insertion. At pH 7.8, the histidines are uncharged and an extended helical conformation including residues 4-21 is again obtained. LAH4 thus exhibits a high degree of conformational plasticity. The structures provide a stroboscopic view of the conformational changes that occur during membrane insertion, and are discussed in the context of antimicrobial activity and DNA transfection.

The heterodimeric antimicrobial peptide distinctin is composed of 2 linear peptide chains of 22-and 25-aa residues that are connected by a single intermolecular S-S bond. This heterodimer has been considered to be a unique example of a previously unrecorded class of bioactive peptides. Here the 2 distinctin chains were prepared by chemical peptide synthesis in quantitative amounts and labeled with 15 N, as well as 15 N and 2 H, at selected residues, respectively, and the heterodimer was formed by oxidation. CD spectroscopy indicates a high content of helical secondary structures when associated with POPC/POPG 3:1 vesicles or in membrane-mimetic environments. The propensity for helix formation follows the order heterodimer >chain 2 >chain 1, suggesting that peptidepeptide and peptide-lipid interactions both help in stabilizing this secondary structure. In a subsequent step the peptides were reconstituted into oriented phospholipid bilayers and investigated by 2 H and proton-decoupled 15 N solid-state NMR spectroscopy. Whereas chain 2 stably inserts into the membrane at orientations close to perfectly parallel to the membrane surface in the presence or absence of chain 1, the latter adopts a more tilted alignment, which further increases in the heterodimer. The data suggest that membrane interactions result in considerable conformational rearrangements of the heterodimer. Therefore, chain 2 stably anchors the heterodimer in the membrane, whereas chain 1 interacts more loosely with the bilayer. These structural observations are consistent with the antimicrobial activities when the individual chains are compared to the dimer. amphipathic alpha-helix ͉ membrane insertion ͉ membrane protein structure determination ͉ peptide-peptide interactions ͉ synergistic activity A s more and more pathogens develop resistance against many commonly used antibiotics, urgent actions are needed to counteract this resistance and new bactericidal and fungicidal compounds have to be developed. Both plants and animals produce, store, and secrete antibiotic peptides in exposed tissues, or synthesize such compounds upon induction, and indeed many antibiotic peptides have been isolated from natural sources (1, 2). The availability of these molecules establishes a defense system that can be set into action immediately when infections occur, and several antimicrobial peptides have been found to also exhibit virucidal and tumorcidal activities (reviewed in refs. 3 and 4). The lack of sequence homology, the activity of chiral analogues, the amphipathic properties, and biophysical measurements all suggest that the bacterial membranes rather than chiral receptors are the targets of many of these peptides (1, 3), although more recent findings suggest that at least some of them also have internal targets after crossing the membrane (5, 6).The heterodimeric peptide distinctin has been isolated from Phyllomedusa distincta, a frog living in the southeast Atlantic forests of Brazil (7). The distinctin is composed of 2 linear peptide chains, one of 22-an...

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy 1H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.