Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by 1 H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 ؎ 9°, making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solventexposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, Pardaxins belong to a class of small amphipathic peptides that form part of the defense mechanism secreted by sole fish of the genus Pardachirus (1). These polypeptides are postulated to be shark-repelling and toxic to several different organisms (2, 3). The physiology and pharmacology of pardaxins is rather complex; their effects range from interference with ionic transport in both the epithelium and nerve cells to morphological changes in the synaptic vesicles of lipid membranes (4 -6). At minimum inhibitory concentrations (3 to 40 M), pardaxins are able to kill bacteria, whereas at higher concentrations (Ͼ50 M), they lyse red blood cell membranes. In addition, pardaxins can disrupt the ionic transport of the osmoregulatory epithelium and presynaptic activity in mammals by forming voltage-dependent and ion-selective channels (1,7,8).An important characteristic of these membrane active peptides is their selective interaction with specific lipid membranes. Several mechanistic studies carried out with synthetic lipids suggest that pardaxins interact with the lipid surface by aggregating and forming pores, and eventually causing leakage of the cellular content (4). The widely accepted mechanism for pardaxin interactions with these membranes is the so-called "barrel-stave" model. This is a multistep mechanism in which the peptides are thought to a) bind the membrane in an ␣-helical structure, b) self-aggregate on the membrane surface, c) insert themselves into the hydrocarbon core of the membrane, and d) recruit more monomers, progressively increasing the size of the pore. Helicity, hydrophobic moment, hydrophobicity, charges, and the angle subtended by the hydrophilic/hydrophobic helix surfaces are all crucial structural parameters that modulate both the activity and selectivity of these membrane active peptides (9, 10).Several biophysical studies show that the known sequences of pardaxins (Fig.

The effect of tricyclic antidepressants (TCA) on phospholipid bilayer structure and dynamics was studied to provide insight into the mechanism of TCA-induced intracellular accumulation of lipids (known as lipidosis). Specifically we asked if the lipid-TCA interaction was TCA or lipid specific and if such physical interactions could contribute to lipidosis. These interactions were probed in multilamellar vesicles and mechanically oriented bilayers of mixed phosphatidylcholine-phosphatidylglycerol (PC-PG) phospholipids using (31)P and (14)N solid-state NMR techniques. Changes in bilayer architecture in the presence of TCAs were observed to be dependent on the TCA's effective charge and steric constraints. The results further show that desipramine and imipramine evoke distinguishable changes on the membrane surface, particularly on the headgroup order, conformation and dynamics of phospholipids. Desipramine increases the disorder of the choline site at the phosphatidylcholine headgroup while leaving the conformation and dynamics of the phosphate region largely unchanged. Incorporation of imipramine changes both lipid headgroup conformation and dynamics. Our results suggest that a correlation between TCA-induced changes in bilayer architecture and the ability of these compounds to induce lipidosis is, however, not straightforward as imipramine was shown to induce more dramatic changes in bilayer conformation and dynamics than desipramine. The use of (14)N as a probe was instrumental in arriving at the presented conclusions.

Characterization of the oligomerization of membrane-associated peptides is important to understand the folding and function of biomolecules like antimicrobial peptides, fusion peptides, amyloid peptides, toxins, and ion channels. However, this has been considered to be very difficult, because the amphipathic properties of the constituents of the cell membrane pose tremendous challenges to most commonly used biophysical techniques. In this study, we present the application of a simple (14)N solid-state NMR spectroscopy of aligned model membranes containing a phosphatidyl choline lipid to investigate the oligomerization of membrane-associated peptides. Since the near-symmetric nature of the choline headgroup of a phosphocholine lipid considerably reduces the (14)N quadrupole coupling, there are significant practical advantages in using (14)N solid-state NMR experiments to probe the interaction of peptide or protein with the surface of model membranes. Experimental results for several membrane-associated peptides are presented in this paper. Our results suggest that the experimentally measured (14)N quadrupole splitting of the lipid depends on the peptide-induced changes in the electrostatic potential of the lipid bilayer surface and therefore on the nature of the peptide, peptide-membrane interaction, and peptide-peptide interaction. It is inferred that the membrane orientation and oligomerization of the membrane-associated peptides can be measured using (14)N solid-state NMR spectroscopy.

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts alpha-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. 2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. 31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from 31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and 31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.

Conformations of the powder samples of poly-l-alanine with molecular weights of 356 Da (tetraalanine), 15 000 Da (PLA-200), and 23 600 Da (PLA-333) were characterized by 13C cross-polarization magic-angle spinning (CPMAS) and 1H combined rotation and multiple pulse (CRAMPS) solid-state NMR spectroscopy. From the 13C and 1H isotropic chemical shift values, it is predicted that the main chain conformations of tetraalanine and PLA-200 are mainly β-sheet while the conformation of PLA-333 is mainly an α-helix. It is unusual and interesting that a high molecular weight homopolypeptide, PLA-200, has a β-sheet conformation rather than an α-helix conformation. The effect of dichloroacetic acid (DCA) solvent on the backbone conformation of these peptides was also studied. It is inferred from solid-state NMR results that conformations of tetraalanine and PLA-333 are similar before and after crystallization from DCA. On the other hand, the backbone conformation of PLA-200 is 60% α-helix and 40% β-sheet after crystallization from the DCA solvent. Variable temperature studies are also reported.