Membrane structure and interactions of human catestatin by multidimensional solution and solid-state NMR spectroscopy.

Catestatin is a natural peptide of higher organisms including humans, with a wide variety of biological functions involved in catecholamine inhibition, cardiovascular regulation, control of blood pressure, inflammation, and innate immunity. It is derived from the natural processing of chromogranin A, induced in the skin after injury, and produced by chromaffin cells and neutrophils. With neutrophils, the peptide enters the cell by crossing the plasma membrane where it interacts with internal targets to induce calcium influx. Therefore, we investigated the membrane interactions and structure of several catestatin-derived peptides. Whereas fluorescence dye release experiments are indicative of membrane permeabilization, multidimensional solution NMR and circular dichroism spectroscopies show that catestatin adopts alpha-helical conformations between Ser-6 and Tyr-12 in the presence of dodecylphosphocholine micelles. Furthermore, proton-decoupled (15)N solid-state NMR spectroscopy of sequences labeled with (15)N and reconstituted into oriented lipid bilayers indicates that this domain is aligned in a strongly tilted to inplanar alignment. Proton-decoupled (31)P NMR spectra of the same samples are indicative of conformational and/or orientational heterogeneity at the level of the lipid bilayer head groups due to the presence of catestatin. The sequence and 3-dimensional structure of catestatin exhibit homologies with penetratin, which is suggestive that they both enter the cells by related mechanisms to target internal structures.

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy.

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 peptide-peptide 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.

Structure and membrane interactions of the antibiotic peptide dermadistinctin K by multidimensional solution and oriented 15N and 31P solid-state NMR spectroscopy.

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an alpha-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with (15)N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting (15)N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled (31)P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.

Solution NMR structures of the antimicrobial peptides phylloseptin-1, -2, and -3 and biological activity: the role of charges and hydrogen bonding interactions in stabilizing helix conformations.

Phylloseptins are antimicrobial peptides of 19-20 residues which are found in the skin secretions of the Phyllomedusa frogs that inhabit the tropical forests of South and Central Americas. The peptide sequences of PS-1, -2, and -3 carry an amidated C-terminus and they exhibit 74% sequence homology with major variations of only four residues close to the C-terminus. Here we investigated and compared the structures of the three phylloseptins in detail by CD- and two-dimensional NMR spectroscopies in the presence of phospholipid vesicles or in membrane-mimetic environments. Both CD and NMR spectroscopies reveal a high degree of helicity in the order PS-2> or =PS-1>PS-3, where the differences accumulate at the C-terminus. The conformational variations can be explained by taking into consideration electrostatic interactions of the negative ends of the helix dipoles with potentially cationic residues at positions 17 and 18. Whereas two are present in the sequence of PS-1 and -2 only one is present in PS-3. In conclusion, the antimicrobial phylloseptin peptides adopt alpha-helical conformations in membrane environments which are stabilized by electrostatic interactions of the helix dipole as well as other contributions such hydrophobic and capping interactions.

Peptide-related alterations of membrane-associated water: deuterium solid-state NMR investigations of phosphatidylcholine membranes at different hydration levels.

Deuterated water associated with oriented POPC bilayers was investigated before and after the addition of 2 mol% peptide. Membranes in the presences of antimicrobial-(LAH4), pore-forming- (the segments M2 of influenza A and S4 of the domain I of rat brain sodium channels) or lysine-containing model peptides (LAK1 and LAK3) were investigated by (2)H and proton-decoupled (31)P solid-state NMR. The NMR spectra were recorded as a function of hydration in the range between 15 and 93% relative humidity and of sample composition. In the presence of peptides an increased association of water is observed. A quantitative analysis suggests that the peptide-induced changes in the lipid bilayer packing have a significant effect on membrane-water association. The quadrupolar splittings of (2)H(2)O at a given degree of hydration indicate that the changes of the water deuterium order parameter are specific for the peptide sequence and the lipid composition.