Lutein and zeaxanthin as protectors of lipid membranes against oxidative damage: the structural aspects.

Two main xanthophyll pigments are present in the membranes of macula lutea of the vision apparatus of primates, including humans: lutein and zeaxanthin. Protection against oxidative damage of the lipid matrix and screening against excess radiation are the most likely physiological functions of these xanthophyll pigments in macular membranes. A protective effect of lutein and zeaxanthin against oxidative damage of egg yolk lecithin liposomal membranes induced by exposure to UV radiation and incubation with 2, 2'-azobis(2-methypropionamidine)dihydrochloride, a water-soluble peroxidation initiator, was studied. Both lutein and zeaxanthin were found to protect lipid membranes against free radical attack with almost the same efficacy. The UV-induced lipid oxidation was also slowed down by lutein and zeaxanthin to a very similar rate in the initial stage of the experiments (5-15 min illumination) but zeaxanthin appeared to be a better photoprotector during the prolonged UV exposure. The decrease in time of a protective efficacy of lutein was attributed to the photooxidation of the carotenoid itself. Both lutein and zeaxanthin were found to slightly modify mechanical properties of the liposomes in a very similar fashion as concluded on the basis of H(1) NMR and diffractometric measurements of pure egg yolk membranes and membranes pigmented with the xanthophylls. Linear dichroism analysis of the mean orientation of the dipole transition moment of the xanthophylls incorporated to the lipid multibilayers revealed essentially different orientation of zeaxanthin and lutein in the membranes. Zeaxanthin was found to adopt roughly vertical orientation with respect to the plane of the membrane. The relatively large orientation angle between the transition dipole and the axis normal to the plane of the membrane found in the case of lutein (67 degrees in the case of 2 mol% lutein in EYPC membranes) was interpreted as a representation of the existence of two orthogonally oriented pools of lutein, one following the orientation of zeaxanthin and the second parallel with respect to the plane of the membrane. The differences in the protective efficacy of lutein and zeaxanthin in lipid membranes were attributed to a different organization of zeaxanthin-lipid and lutein-lipid membranes.     

Macromolecular crowding at membrane interfaces: adsorption and alignment of membrane peptides

Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH₄, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, β-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and β-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (> 85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin

The mechanism of membrane interaction of two amphipathic antimicrobial peptides, MSI-78 and MSI-594, derived from magainin-2 and melittin, is presented. Both the peptides show excellent antimicrobial activity. The 8-anilinonaphthalene-1-sulfonic acid uptake experiment using Escherichia coli cells suggests that the outer membrane permeabilization is mainly due to electrostatic interactions. The interaction of MSI-78 and MSI-594 with lipid membranes was studied using 31P and 2H solid-state NMR, circular dichroism, and differential scanning calorimetry techniques. The binding of MSI-78 and MSI-594 to the lipid membrane is associated with a random coil to alpha-helix structural transition. MSI-78 and MSI-594 also induce the release of entrapped dye from POPC/POPG (3:1) vesicles. Measurement of the phase-transition temperature of peptide-DiPoPE dispersions shows that both MSI-78 and MSI-594 repress the lamellar-to-inverted hexagonal phase transition by inducing positive curvature strain. 15N NMR data suggest that both the peptides are oriented nearly perpendicular to the bilayer normal, which infers that the peptides most likely do not function via a barrel-stave mechanism of membrane-disruption. Data obtained from 31P NMR measurements using peptide-incorporated POPC and POPG oriented lamellar bilayers show a disorder in the orientation of lipids up to a peptide/lipid ratio of 1:20, and the formation of nonbilayer structures at peptide/lipid ratio>1:8. 2H-NMR experiments with selectively deuterated lipids reveal peptide-induced disorder in the methylene units of the lipid acyl chains. These results are discussed in light of lipid-peptide interactions leading to the disruption of membrane via either a carpet or a toroidal-type mechanism.     

PARP-1 regulates the expression of caspase-11

Lariciresinol is an enterolignan precursor isolated from the herb Sambucus williamsii, a folk medicinal plant used for its therapeutic properties. In this study, the antifungal properties and mode of action of lariciresinol were investigated. Lariciresinol displays potent antifungal properties against several human pathogenic fungal strains without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of action of lariciresinol, the membrane interactions of lariciresinol were examined. Fluorescence analysis using the membrane probe 3,3′-diethylthio-dicarbocyanine iodide (DiSC₃-5) and 1,6-diphenyl-1,3,5-hexatriene (DPH), as well as a flow cytometric analysis with propidium iodide (PI), a membrane-impermeable dye, indicated that lariciresinol was associated with lipid bilayers and induced membrane permeabilization. Therefore, the present study suggests that lariciresinol possesses fungicidal activities by disrupting the fungal plasma membrane and therapeutic potential as a novel antifungal agent for the treatment of fungal infectious diseases in humans.     

Location of the bioactive pentacyclic triterpene ursolic acid in the membrane. A molecular dynamics study

Ursolic acid (URS), an ursane-representative bioactive pentacyclic triterpene, is a plant secondary metabolite presenting a great number of pharmacological beneficial properties. Due to the prominent hydrophobic character of URS and its high phospholipid/water partition coefficient, some of its possible effects on biological systems might be related to its capacity to interact with and locate into the membrane as well as interact specifically with its components. In this work, we have studied the location and orientation of URS in the membrane by molecular dynamics simulations. At the end of the simulation, URS locates near the surface in vicinity to the phospholipid headgroups but its orientation depends on lipid composition, its final average orientation being a nearly parallel one in POPC but a nearly perpendicular one in POPC/POPE/POPG/PSM/Chol. Furthermore, in the complex lipid system URS seems to interact specifically with POPE, PSM, and Chol excluding POPG from its surroundings, which could lead to phase separation and domain formation. The different disposition of URS in the membrane and its specific interaction with certain lipid types could lead to a significant perturbation of the membrane structure. The important pharmacological activities of URS would rely on the effects it exerts on the membrane structure in general and the existence of specific interactions with specific lipids in particular.     

ENaC-membrane interactions: regulation of channel activity by membrane order

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459-6463). As these changes of temperature affect membrane lipid order and lipid-protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order-dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2 degrees C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.     

Organisation of xanthophyll pigments lutein and zeaxanthin in lipid membranes formed with dipalmitoylphosphatidylcholine

Carotenoid pigments and in particular xanthophylls play several physiological functions in plant and animal membranes. Xanthophylls are present in biological membranes in the form of pigment-protein complexes but also as direct components of lipid phase. The biological activity of carotenoids in membranes depends on a molecular organisation of pigments in lipid bilayers, in particular the localisation, orientation and aggregational state. In the present work the organisation of lutein- and zeaxanthin-containing lipid membranes was analysed with the application of electronic absorption spectroscopy. Both xanthophyll pigments incorporated to the dipalmitoylphosphatidylcholine (DPPC) unilamellar liposomes form H-type molecular aggregates, manifested by the hypsochromic shift of the main absorption band of carotenoids. The aggregation of lutein and zeaxanthin in DPPC membranes was observed even at relatively low concentrations of a pigment in the lipid phase (1-5 mol%). Gaussian analysis of the absorption spectra of lutein and zeaxanthin in DPPC membranes in terms of the exciton splitting theory revealed the formation of different molecular structures of pigments interpreted as dimers, trimers, tetramers and large aggregates. The fraction of lutein and zeaxanthin in the monomeric form was found to depend on the physical state of the lipid phase. Pronounced monomerisation of lutein and zeaxanthin was observed as accompanying the transition from the P(beta)' phase to the L(alpha) phase of DPPC, mostly at the expense of the trimeric and tetrameric forms. The fraction of monomers of lutein is always lower by 10-30% than that of zeaxanthin under the same experimental conditions. Different organisational forms of lutein and zeaxanthin in the model system studied are discussed in terms of possible physiological functions of these pigments in the membranes of the retina: zeaxanthin in the protection of the lipid phase against oxidative damage and lutein in absorbing short wavelength radiation penetrating retina membranes.     

Mode of insertion of chlorophyll a in a lipid layer. A conformational approach

A new procedure for conformational analysis is described to define the orientation of chlorophyll a in model membranes. The method allows to predict the configuration of assembled amphiphilic molecules. The minimal conformational energy is calculated as the sum of the contributions, resulting from the Van der Waals interactions, the torsional potentials, the electrostatic interactions and the transfer energy. In the calculated conformation, the porphyrin ring is orientated at an angle of 45° ± 5° to the membrane interface, with the phytol chain inserted into the lipid layer. This orientation of the porphyrin ring is in excellent agreement with the experimental value obtained with model membranes. The method could allow to define the orientation of other components in the thylakoid membrane.     

Dipalmitoylphosphatidylcholine membranes modified with zeaxanthin: numeric study of membrane organisation

The model of a dipalmitoylphosphatidylcholine (DPPC) bilayer containing a xanthophyll pigment zeaxanthin (ZEA) is proposed. The model is based on the ten-state Pink-Green-Chapman model of a lipid monolayer. The Monte Carlo method of computer simulation has been applied. Our model of the lipid membrane consists of two lipid monolayers with ZEA molecules spanning the lipid bilayer. The concentration of ZEA molecules is assumed to be conserved. Within the model, the interactions between lipid monolayers in a bilayer exist through ZEA molecules only. The experimental data concerning the aggregation of ZEA in DPPC from the literature and from our research were applied as a criterion to fit the model parameters. The model gives the dependences of the main phase transition temperature on ZEA/DPPC molar ratio, the percentage of ZEA in a monomeric form on ZEA/DPPC molar ratio and on temperature. The dependences obtained within the model and the experimental ones are in qualitative agreement. The influence of intermolecular interaction parameters on ZEA aggregation has been discussed. The differences between the model and the experimental results concerning mainly the pattern of ZEA aggregation have been discussed. Analyses of the lipid microconfiguration allow to advance the hypothesis concerning the influence of ZEA on the membrane permeability.     

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein–membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H₀) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed.     

Solid-state NMR spectroscopy to study protein–lipid interactions

The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid–protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid–protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid–protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K⁺ channel KcsA are discussed. This article is part of a Special Issue entitled Tools to study lipid functions.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Deciphering Membrane Insertion of the Diphtheria Toxin T Domain by Specular Neutron Reflectometry and Solid-State NMR Spectroscopy

Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane.     

Bicelles: A natural ‘molecular goniometer’ for structural,dynamical and topological studies of molecules in membranes

Major biological processes occur at the biological membrane. One of the great challenges is to understand the function of chemical or biological molecules inside the membrane; as well of those involved in membrane trafficking. This requires obtaining a complete picture of the in situ structure and dynamics as well as the topology and orientation of these molecules in the membrane lipid bilayer. These led to the creation of several innovative models of biological membranes in order to investigate the structure and dynamics of amphiphilic molecules, as well as integral membrane proteins having single or multiple transmembrane segments. Because the determination of the structure, dynamics and topology of molecules in membranes requires a macroscopic alignment of the system, a new membrane model called ‘bicelles’ that represents a crossover between lipid vesicles and classical micelles has become very popular due to its property of spontaneous self-orientation in magnetic fields. In addition, crucial factors involved in mimicking natural membranes, such as sample hydration, pH and salinity limits, are easy to control in bicelle systems. Bicelles are composed of mixtures of long chain (14–18 carbons) and short chain phospholipids (6–8 carbons) hydrated up to 98% with buffers and may adopt various morphologies depending on lipid composition, temperature and hydration. We have been developing bicelle systems under the form of nano-discs made of lipids with saturated or biphenyl-containing fatty acyl chains. Depending on the lipid nature, these membranous nano-discs may be macroscopically oriented with their normal perpendicular or parallel to the magnetic field, providing a natural ‘molecular goniometer’ for structural and topological studies, especially in the field of NMR. Bicelles can also be spun at the magic angle and lead to the 3D structural determination of molecules in membranes.     

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X = 0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Lipid-Modulated Sequence-Specific Association of Glycophorin A in Membranes

Protein association in lipid membranes is a complex process with thermodynamics directed by a multitude of different factors. Amino-acid sequence is a molecular parameter that affects dimerization as shown by limited directed mutations along the transmembrane domains. Membrane-mediated interactions are also important although details of such contributions remain largely unclear. In this study, we probe directly the free energy of association of Glycophorin A by means of extensive parallel Monte Carlo simulations with recently developed methods and a model that accounts for sequence-specificity while representing lipid membranes faithfully. We find that lipid-induced interactions are significant both at short and intermediate separations. The ability of molecules to tilt in a specific hydrophobic environment extends their accessible interfaces, leading to intermittent contacts during protein recognition. The dimer with the lowest free energy is largely determined by the favorable lipid-induced attractive interactions at the closest distance. Finally, the coarse-grained model employed herein, together with the extensive sampling performed, provides estimates of the free energy of association that are in excellent agreement with existing data.