Peptide conformational changes induced by tryptophan-phosphocholine interactions in a micelle

Sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles are often used to mimic the membrane- or receptor-bound states of peptides in NMR studies. From the present examination of a 26-residue analog of exendin-4 (TrEX4) by NMR and CD in water, aqueous 30% trifluoroethanol (TFE), and bound to both SDS and DPC micelles, it is clear that these two lipid micelles can yield very different peptide structures. The Trp-cage fold (also observed in 30% TFE) is present when TrEX4 is bound to SDS micelles; however, tertiary structure is absent in the presence of DPC micelles. The loss of tertiary structure is attributed to an energetically favorable interaction (estimated as 2-3 kcal/mol) of the tryptophan side chain with the phosphocholine head groups. These dramatic structural differences suggest that care must be taken when using either SDS or DPC to mimic the membrane- or receptor-bound states.     

Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).     

Recovery of protein from reverse micelles through gas hydrate formation

The recovery of cytochrome c and ribonuclease A from di-2-ethylhexyl sodium sulfosuccinate (AOT) reverse micelles have been examined by the gas hydrate formation. The recovery of cytochrome c depended upon the kind of gas and the water content (w0=[H₂O]/[AOT]) of reverse micellar solution containing cytochrome c prepared. Recoveries of cytochrome c and ribonuclease A were more than 80%, when 1,1,1,2-tetrafluoroethane (TFE) was used as a hydrating gas. The activity of cytochrome c recovered from reverse micelles was maintained perfectly.     

FTIR study of horseradish peroxidase in reverse micelles

Fourier transform infrared (FTIR) method was used to study the secondary structures of horseradish peroxidase (HRP) in aqueous solution and in reverse micelles for the first time. Results indicated that the structure of HRP in sodium bis(2-ethylhexy)sulfosuccinate (AOT) reverse micelles was close to that in aqueous solution. In cetyltrimethylammonium bromide (CTAB) and sodium dodecylfate (SDS) reverse micelles the position of some bands changed. Results indicated that the secondary structure had a close relationship with the surfactant species of the reverse micelles. Among the three types of reverse micelles, the system of AOT reverse micelles was probably the most beneficial reaction media to HRP.     

Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes

Nramp1 (natural resistance-associated macrophage protein 1) is an integral membrane protein with 12 putative transmembrane domains. As a proton-coupled divalent metal cation transporter, it is involved in defense against intracellular pathogens. Disease-causing mutation in Nramp1 occurring at glycine 169 located within the fourth transmembrane domain (TM4) suggests functional importance of this domain. In this paper, we study the three-dimensional structures of a peptide, corresponding to the TM4 of the wild-type Nramp1, in SDS micelles and 2, 2, 2-trifluoroethanol solvent using CD and NMR spectroscopies. We have found that an alpha-helix is predominantly induced in membrane-mimetic environments and the folding of the C-terminal residues is regulated by pH in SDS micelles. The peptide is embedded in SDS micelles and self-associated by coiled-coil interactions. The helix of the peptide in TFE is lengthened towards the N-terminus compared with those in SDS micelles at acidic pH and the self-association of the peptide is also observed in TFE. The fact that Mn(2+) ions are accessible to Asp-14 located in the interior of SDS micelles is found and the binding affinity is increased with increasing pH. The self-association of the peptide may provide a path by which Mn(2+) ions pass through the membrane.     

Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes

Nramp1 (natural resistance-associated macrophage protein 1) is an integral membrane protein with 12 putative transmembrane domains. As a proton-coupled divalent metal cation transporter, it is involved in defense against intracellular pathogens. Disease-causing mutation in Nramp1 occurring at glycine 169 located within the fourth transmembrane domain (TM4) suggests functional importance of this domain. In this paper, we study the three-dimensional structures of a peptide, corresponding to the TM4 of the wild-type Nramp1, in SDS micelles and 2, 2, 2-trifluoroethanol solvent using CD and NMR spectroscopies. We have found that an α-helix is predominantly induced in membrane-mimetic environments and the folding of the C-terminal residues is regulated by pH in SDS micelles. The peptide is embedded in SDS micelles and self-associated by coiled-coil interactions. The helix of the peptide in TFE is lengthened towards the N-terminus compared with those in SDS micelles at acidic pH and the self-association of the peptide is also observed in TFE. The fact that Mn²⁺ ions are accessible to Asp-14 located in the interior of SDS micelles is found and the binding affinity is increased with increasing pH. The self-association of the peptide may provide a path by which Mn²⁺ ions pass through the membrane.     

Extraction of bovine serum albumin using nanoparticulate reverse micelles

The extraction of a relatively large molecular weight protein, bovine serum albumin (BSA), using nano-sized reverse micelles of nonionic surfactant polyoxyethylene p-t-octylphenol (Triton-X-100) is attempted for the first time. Suitability of reverse micelles of anionic surfactant sodium bis (2-ethyl hexyl) sulfosuccinate (AOT) and Triton-X-100/AOT mixture in organic solvent toluene for BSA extraction is also investigated. Although, the size of the Triton-X-100 reverse micelle in toluene is large enough to host BSA molecule in the hydraulic core, the overall extraction efficiency is found to be low, which may be due to lack of strong driving force. AOT/toluene system resulted in complete forward extraction at aqueous pH 5.5 and a surfactant concentration of 160 mM. The back extraction with aqueous phase (pH 5.5) resulted in 100% extraction of BSA from the organic phase. The addition of Triton-X-100 to AOT reduced the extraction efficiency of AOT reverse micelles, which may be attributed to reduced hydrophobic interaction. The circular dichroism (CD) spectrum of BSA extracted using AOT/toluene reverse micelles indicated the structural stability of the protein extracted.     

Solubilization of soybean mitochondria in AOT/isooctane water-in-oil microemulsions

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30-40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Structure analysis of a fusogenic peptide sequence from the sea urchin fertilization protein bindin

The structure of "B18", an 18-residue fusogenic peptide from the sea urchin fertilization protein bindin, was investigated in several membrane-mimicking environments with circular dichroism and nuclear magnetic resonance spectroscopy. The fully conserved peptide sequence represents the minimal functional part of the 24 kDa protein, which can bind to membranes and induce fusion of lipid vesicles. The B18 peptide undergoes a coil-helix transition in the presence of TFE, showing a transient tendency to self-associate. Its NMR structure in 30% TFE exhibits two helical regions at either side, connected by a flexible loop. In DPC and SDS detergent micelles, this loop becomes distinctly bent, presumably due to the high degree of curvature of the micelles. The loop contains a histidine-rich motif for binding zinc, which is required for the fusogenic function of the peptide. Therefore, we monitored the structural response of B18 and of recombinant bindin toward this ion. Like TFE, and in a mutually cooperative manner, zinc induces a partially helical structure in both the peptide and the protein. Complex formation via the histidine residues rigidifies the flexible loop and is accompanied by self-association of the molecules. The data suggest that the zinc-bound functional state is a continuous amphipathic alpha-helix, bearing some resemblance to a leucine zipper. Two hydrophobic patches on one face could favorably penetrate into a membrane, while two arginines on the other face could interact with lipid phosphate groups. The three-dimensional model of the B18 sequence thus contributes to a better understanding of peptide-induced vesicle fusion in general, and of the lipid-protein interactions of sperm bindin in particular.     

2D-NMR and ATR-FTIR study of the structure of a cell-selective diastereomer of melittin and its orientation in phospholipids

Melittin, a 26 residue, non-cell-selective cytolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V(5,8),I(17),K(21)-melittin, D-amino acids at positions V(5,8),I(17),K(21)) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V(5,8),I(17),K(21)-melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]-V(5,8),I(17),K(21)-melittin binds strongly and destabilizes only negatively charged phospholipid vesicles, in contrast to native melittin, which binds strongly also zwitterionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D]-V(5,8),I(17),K(21)-melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were done with both melittin and [D]-V(5,8), I(17),K(21)-melittin. The structure of the diastereomer was characterized by NMR in water, as well as in three different membrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, methanol, and dodecylphosphocholine/phosphatidylglycerol (DPC/DMPG) micelles. The NMR data revealed an amphipathic alpha-helix only in the C-terminal region of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V(5,8),I(17),K(21)-melittin are oriented parallel to the membrane surface. This study indicates the role of secondary structure formation in selective cytolytic activity of [D]-V(5,8), I(17),K(21)-melittin. While the N-terminal helical structure is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural element for binding and insertion into zwitterionic membranes and for hemolytic activity.     

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata.

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.     

Conformational studies of human [15-2-aminohexanoic acid]little gastrin in sodium dodecyl sulfate micelles by 1H NMR

Human little gastrin is a 17 amino acid peptide that adopts a random conformation in water and an ordered structure in sodium dodecyl sulfate (SDS) micelles as well as in trifluoroethanol (TFE). The circular dichroism spectra in these two media have the same shape, indicative of a similar preferred conformation [Mammi, S., Mammi, N. J., Foffani, M. T., Peggion, E., Moroder, L., & Wünsch, E. (1987) Biopolymers 26, S1-S10]. We describe here the assignment of the proton NMR resonances and the conformational analysis of [Ahx15]little gastrin in SDS micelles. Two-dimensional correlation techniques form the basis for the assignment. The conformational analysis utilized NOE's, NH to C alpha H coupling constants, and the temperature coefficients of the amide chemical shifts. The NMR data indicate a helical structure in the N-terminal portion of the peptide. These results are compared with the conformation that we recently proposed for a minigastrin analogue (fragment 5-17 of [Ahx15]little gastrin) in TFE.     

Preferred conformation of endomorphin-1 in aqueous and membrane-mimetic environments.

The newly discovered endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are potent opioid peptides with the highest affinity and selectivity for the mu receptor among all known endogenous ligands. To investigate a possible correlation between these biological properties and the conformational preferences of the small peptides, a comparative structural analysis was performed of endomorphin-1 in aqueous buffer and in membrane-mimicking SDS and AOT normal and reverse micelles by the use of CD, FT-IR, fluorescence and(1)H-NMR spectroscopy. It is well established for opioid peptides that, independently of the receptor selectivity, the Tyr1 residue plays the role of the primary pharmacophore and that the orientation of the second aromatic pharmacophore relative to the tyrosine side-chain dictates the mu or delta-receptor selectivity. By varying the environment of endomorphin-1 from water to the amphipathic SDS micelles and even more efficiently to the AOT reverse micelles, the display of the aromatic side-chains changes from an interaction of the Tyr1 and Phe4 residues to a switch of the Trp3 indole group into close contact with the phenolic moiety to prevent this type of interaction and to force an orientation of the Phe4 side-chain into the opposite direction. This conformational switch is accompanied by a stabilization of the cis -Pro2 isomer and the resulting spatial array of the pharmacophoric groups correlate well with the structural model of mu receptor-bound opioid peptides. The results indicate that AOT reverse micelles with a woof 10, where almost exclusively ordered water is secluded in the cavity, constitute with their electrostatic and hydrophobic potential an excellent mimetic of amphipathic surfaces as present on lipid bilayers and on ligand-recognition and ligand-binding sites of proteins.     

Interactions of the antimicrobial peptide Ac-FRWWHR-NH(2) with model membrane systems and bacterial cells.

The acetylated and amidated hexapeptide FRWWHR (combi-2), previously identified by combinatorial chemistry methods, shows strong antimicrobial activity. The binding of the peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-[(phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles was performed to determine changes in the lipid phase behaviour upon binding the peptide. Two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy, to solve the bound peptide structure, was performed in the presence of dodecylphosphatidylcholine (DPC) and sodium dodecyl sulphate (SDS) micelles. The fluorescence, ITC and DSC studies indicate that the peptide interacts preferentially with lipid vesicles containing negatively charged head groups. Conformational information determined using NMR indicate that the combi-2 peptide adopts a coiled amphipathic conformation when bound to SDS and DPC micelles. Leakage assays indicate that the peptide is not very efficient at causing leakage from calcein-filled large unilamellar vesicles comprised of POPG/POPC (1 : 1). The rapid passage of either the fluorescent-tagged peptides combi-2 or the previously studied peptide Ac-RRWWRF-NH(2) (combi-1) into Escherichia coli and Staphylococcus aureus suggests that instead of membrane disruption, the main bactericidal site of action of these peptides might be located inside bacteria.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Synthesis and secondary structure of loop 4 of myelin proteolipid protein: effect of a point mutation found in Pelizaeus-Merzbacher disease.

To study the effects of a point mutation found in Pelizaeus-Merzbacher disease (PMD) on the physicochemical and structural properties of the extracellular loop 4 of the myelin proteolipid protein (PLP), we synthesized the peptide PLP(181-230)Pro215 and one mutant PLP(181-230)Ser215 with regioselective formation of the two disulphide bridges Cys200-Cys219 and Cys183-Cys227. As conventional amino acid building blocks failed to give crude peptides of good quality we had to optimize the synthesis by introducing pseudoproline dipeptide building blocks during the peptide elongation. In peptide Pro215 the first bridge Cys200-Cys219 was obtained after air oxidation, but in peptide Ser215 because of aggregation, dimethyl sulfoxide (DMSO) oxidation had to be used. The second bridge Cys183-Cys227 was obtained by iodine oxidation of both Cys (acetamidomethyl, Acm)-protected peptides. The secondary structures of the parent and mutant loops were analysed by circular dichroism (CD) in the presence of trifluoroethanol (TFE) and sodium dodecyl sulphate (SDS) as a membrane mimetic. Analysis of the spectra showed that the content of alpha-helix and beta-sheet varied differently for both peptides in TFE and SDS solutions, demonstrating the sensitivity of their conformation to the environment and the differences in their secondary structure. The ability of both peptides to insert into the SDS micelles was assayed by intrinsic tryptophan fluorescence.     

Structure of an analog of fusion peptide from hemagglutinin

A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0-6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH < or = 6 and the helical content in the peptide increased in the row: water < water/TFE < DPC approximately = phospholipid vesicle while the amount of beta-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic alpha-helix in residues 2-18, which is flexible in 11-18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa approximately 5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) deltaCOO- (H+)...HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is approximately 5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).     

A synthetic analog of plantaricin 149 inhibiting food-borne pathogenic bacteria:evidence for alpha-helical conformation involved in bacteria-membrane interaction.

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane.Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014.The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.