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ABSTRACT: This work focuses on the adsorption kinetics of dicynthaurin with lipid monolayers, the effect of peptide adsorption on the structure of the lipid condensed chain lattice, peptide orientation and secondary structure in the adsorbed state. The studies with DPPG as model system revealed strong adsorption and massive incorporation of the peptide into the monolayer. Infrared reflection absorption spectroscopy (IRRAS) experiments showed that the secondary structure of the peptide is maintained upon adsorption. Specular X-ray reflectivity showed the destabilization of the condensed phase of the pure lipid monolayer and revealed a tilted orientation of the long axis of the peptide helix of about 40 degrees from the surface normal. Incorporation of the peptide was found to be pressure dependent, and at high pressure a "squeeze-out" was observed; however, the peptide remained localized to the interface, as suggested by infrared data. These findings were supported by optical fluorescence microscopy measurements which showed the squeeze-out of the peptide on water, but not under physiological conditions. The results suggest that dicynthaurin is able to adsorb to the phosphatidylglycerol-rich inner cytoplasmic membrane of bacteria and alter membrane integrity. To identify and interact with membrane motifs that are characteristic of microbes, but which are absent in eukaryotic cells, might be an intrinsic ability of peptide antibiotics.
ChemBioChem 07/2007; 8(9):1038-47. · 3.94 Impact Factor
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ABSTRACT: The interaction of the antimicrobial peptide dicynthaurin (ala) monomer with model membranes of zwitterionic and negatively charged lipids and mixtures thereof was studied by means of isothermal titration calorimetry (ITC), fluorescent leakage, and dynamic light scattering (DLS) measurements. For the ITC analysis, we have applied the surface partitioning equilibrium model which shows that the interaction is predominately driven by hydrophobic effects (Kb between 2 x 10(4) and 1 x 10(5) M(-1)). Under low salt conditions, the enhanced electrostatic interaction leads to larger peptide concentrations immediately above the vesicle surface, which initiates the insertion of the peptide into the bilayer more effectively. Fluorescent leakage measurements have shown a fast leakage of the fluorescent dye within seconds after peptide addition. The analysis of the leakage kinetics was performed in terms of an initial pore formation model (up to t = 1000 s) that takes the reversible surface aggregation of bound peptide monomers into account. From this analysis, a minimum aggregation number of n = 7 +/- 2 per pore is obtained.
The Journal of Physical Chemistry B 07/2007; 111(22):6280-7. · 3.70 Impact Factor
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ABSTRACT: This paper is focused on the thermodynamics and the structural investigation of the interaction of the antimicrobial peptide dicynthaurin monomer with model lipid membranes composed of mixtures of 1-palmitoyl-2-oleyl-glycerophosphocholine and -glycerophosphoglycerol. The thermodynamic binding parameters as obtained by isothermal titration calorimetry reveal strong binding toward the lipid model system dominated by large chemical binding constants which exceeds the electrostatic binding effects and thus suggests insertion of the amphipathic alpha-helical peptide into the hydrophobic membrane core. Circular dichroism study shows that the peptide exhibits trans-membrane alpha-helix secondary structure. Neutron diffraction measurements using partially deuterated sequences were successfully applied to determine the orientation of the peptide thus proving insertion into the hydrophobic membrane core. This insertion and the formation of higher order porelike aggregates is assumed to be the most relevant event in microbial membrane perturbation that in vivo finally leads to bacterial cell death on a fast time scale.
Biochemistry 06/2007; 46(19):5678-86. · 3.42 Impact Factor
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ABSTRACT: This paper reports the first study on the interaction of the antimicrobial peptide dicynthaurin with 1,2-dipalmitoyl-glycerophosphatidyl-glycerol investigated in monolayers at the air-liquid interface. The influence of the peptide on the two-dimensional phase behavior of the negatively charged lipid was elucidated by means of pressure-area isotherm measurements, fluorescence microscopy, and grazing incidence X-ray diffraction measurements. The pure peptide forms a stable monolayer at the air-liquid interface up to 30 mN/m as shown for both the monomeric and the dimeric cynthaurins. The peptide lipid interaction was monitored in isotherm measurements showing a strong adsorption of the peptide and stabilization at the interface promoted by the lipid monolayer. The X-ray diffraction measurements in agreement with fluorescence microscopy studies showed that the peptide destabilizes the condensed chain lattice, leading to a complete fluidization of the condensed lipid phase on physiological buffer. The adsorption of the peptide to the negatively charged lipid monolayer and the fluidization of the condensed chain lattice suggest a direct link to the peptides' ability to expand the bacterial membrane that would be relevant for the in vivo mode of action.
The Journal of Physical Chemistry B 05/2007; 111(14):3813-21. · 3.70 Impact Factor
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ABSTRACT: The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding.
European Biophysics Journal 05/2007; 36(4-5):491-8. · 2.14 Impact Factor
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ABSTRACT: In this work, we report on recent investigations, both on the global and on the local molecular architecture of supported phospholipid model membranes. A brief theoretical introduction explains how global structural information on supramolecular lipid ensembles can be retrieved from surface X-ray diffraction measurements as well as how spin-label electron paramagnetic resonance spectroscopy (EPR) provides complementary information on the local environment of probe molecules. The combination of especially designed X-ray cells with the technique of small- and wide-angle X-ray surface scattering makes it possible to explore various fields of lipid research and its applications. Examples for different physico-chemical conditions are presented: (i) in situ chemistry under excess of water conditions demonstrating how solid-supported lipid films sense salinity, (ii) the 3D electron density reconstruction of a vesicle-fusion intermediate under controlled humidity, and (iii) complementary temperature and pressure effects on oriented phospholipid samples. Further, special attention has been given to the influence of different film preparation techniques with respect to quality and the defect structure manifestation. To resolve the proportions and local properties of defects in a hydrated lipid-deposited surface, spin-label EPR was applied. The results from 9.6 GHz EPR as well as from 1.2 GHz EPR suggest the alignment to be in the range between 30% and 80%. In addition, slow time-dependent EPR measurements point to nano-structural rearrangements due to water flow and reduction of alignment quality.
Advances in Colloid and Interface Science 12/2004; 111(1-2):63-77. · 8.12 Impact Factor
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ABSTRACT: The influence of LiCl solutions on liposomal and surface-supported phosphatidylcholine/water systems (dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), respectively) has been studied by small-angle X-ray techniques. In liposomal dispersions of DPPC, an osmotically stressed liquid-crystalline phase, denoted as Lalpha osm, forms readily after rapid mixing with salt solutions. The transition from Lalpha -->Lalpha osm proceeds in two steps. The first step takes place within seconds and is due to water diffusion from the liposome into the bulk solution. The second, slower process (minutes) can be attributed to the relaxation of initially deformed intermediate liposomes into spherical ones. In experiments with aligned lipid bilayers supported on silicon wafers, it was possible to reproducibly exchange different concentrations of LiCl solutions on a single sample and to determine the lattice changes by time-resolved X-ray scattering at grazing incidence. Independently of the deposition technique (spray- or spin-coating, respectively), none of the investigated POPC samples displayed an osmotically stressed liquid-crystalline phase. While liposomes can be considered nearly defect-free, supported bilayer stacks show a high abundance of defects, such as oily streaks typical of the Lalpha phase. Thus, the alkali ions are free to diffuse into the interbilayer water regions and to cause a slight increase of the bilayer separation (about 1 Angstroms). It is concluded that low to medium concentrations of Li+ ions partially screen the attractive van der Waals force between adjacent membrane layers. However, upon annealing the defect regions or regions of high curvature in the oriented lipid matrix, e.g. by low amounts of oleyl alcohol (OA), the system is able to sense osmotic stress upon addition of a salt solution.
Langmuir 06/2004; 20(11):4621-8. · 4.19 Impact Factor
