Lipid-polypeptide interactions in bilayer lipid membranes

Summary The modifications of the electrical properties of bilayer lipid membranes (BLM) composed of cholesterol and an ionic surfactant upon interaction with charged polypeptides were studied. The addition of 10^–8 m polylysine (Ps⁺) to one side of anionic cholesterol dodecylphosphate BLM increases the specific membrane conductance over 1000-fold (from 10^–8 to 10^–5 mho/cm²) and develops a cationic transmembrane potential larger than 50 mV. This potential is reverted by addition of polyanions such as RNA, polyglutamic or polyadenilic acid to the same side on which Ps⁺ is present, by addition of Ps⁺ to the opposite side, or by addition of trypsin to either side. Both conductance and potential changes are hindered by increasing the ionic strength or by raising the pH of the bathing medium, disappearing above pH 11.5 where it is known that Ps⁺ folds into an -helix. The interaction of polyglutamic acid (PGA) with a cationic cholesterol-hexadecyltrimethylammonium bromide BLM results in increased membrane conductance and development of an anionic transmembrane potential which is reverted by addition of polycations to the same aqueous phase where PGA is present. Addition of either Ps⁺ or PGA to one or both sides of a neutral BLM composed of 7-dehydrocholesterol induces no significant change. The observations suggest the formation of a lipid polymer membrane resultant from the interaction, predominantly electrostatic, of the isolated components. The implications of these results are discussed in terms of the current models of membrane structure.     

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Implicit solvent simulations of peptide interactions with anionic lipid membranes

A recently developed implicit membrane model (IMM1) is supplemented with a Gouy-Chapman term describing counterion-screened electrostatic interactions of a solute with negatively charged membrane lipids. The new model is tested on peptides that bind to anionic membranes. Pentalysine binds just outside the plane of negative charge, whereas Lys-Phe peptides insert their aromatic rings into the hydrophobic core. Melittin and magainin 2 bind more strongly to anionic than to neutral membranes and in both cases insert their hydrophobic residues into the hydrocarbon core. The third domain of Antennapedia homeodomain (penetratin) binds as an alpha-helix in the headgroup region. Cardiotoxin II binds strongly to anionic membranes but marginally to neutral ones. In all cases, the location and configuration of the peptides are consistent with experimental data, and the effective energy changes upon binding compare favorably with experimental binding free energies. The model opens the way to exploring the effect of membrane charge on the location, conformation, and dynamics of a large variety of biologically active peptides on membranes.     

Interaction of glucagon with artificial lipid bilayer membranes.

The enhancement of fluorescence emission from the tryptophan residue of glucagon, the quenching of that emission with acrylamide and with 5-doxyl and 16-doxyl stearic acid, circular dichroism spectra, the release of 6-carboxyfluorescein, and polarized infrared attenuated total reflection (IR-ATR) spectra were used to study the interaction of glucagon with intact lipid vesicles and flat bilayers. Dimyristoylphosphatidylcholine bound the peptide only below the main transition temperature, thus confirming earlier results of Epand et al. (1977). However, the peptide is also bound by vesicles of unsaturated lipids above their transition temperature, suggesting an influence of lipid area on the binding process. Circular dichroism showed that binding to such vesicles also increases the helix content of glucagon. The IR-ATR study and a comparison with dynorphin-A-(1-13)-tridecapeptide revealed profound differences in orientation of the two peptides. The dichroic ratios and the derived order parameters indicated an isotropic orientation of the helical segments of glucagon, but did not exclude a principal orientation of the molecules lying flat on the membrane surface. In contrast, the axis of the dynorphin helix is clearly oriented normal to the interface. The two peptides also differ in their rates of 6-carboxyfluorescein release, suggesting a deeper penetration of the primary amphiphilic helix of dynorphin A-(1-13) than of the secondary amphiphilic helix of glucagon.     

Secondary structure of a mitochondrial signal peptide in lipid bilayer membranes

The secondary structure of the synthetic signal peptide of cytochrome c oxidase subunit IV (coxIV-25) has been measured by circular dichroism spectroscopy in different lipid environments. CoxIV-25 is polymorphic in membranes. It forms an amphiphilic alpha-helix both in negatively charged lipid bilayers (up to 49% helix) and in detergent micelles (up to 42% helix). In association with bilayers of the zwitterionic lipid phosphatidylcholine, coxIV-25 takes an aperiodic, unidentified structure. CoxIV-25 is also partially alpha-helical in bilayers of cardiolipin, mitochondrial lipid extracts and mixtures of synthetic phosphatidylcholine and phosphatidylglycerol.     

Ionic transport in lipid bilayer membranes.

The current-voltage relationships of model bilayer membranes have been measured in various phospholipid systems, under the influence of both a gradient of potential and an ionic concentration, in order to describe the ion translocation through hydrated transient defects (water channels) across the bilayer formed because of lipid structure fluctuations and induced by temperature. The results have been analyzed in the light of a statistical rate theory for the transport process across a lipid bilayer, recently proposed by Skinner et al. (1993). In order to take into account the observed I-V curves and in particular the deviation from an ohmic behavior observed at high potential values, the original model has been modified, and a new version has been proposed by introducing an additional kinetic process. In this way, a very good agreement with the experimental values has been obtained for all of the systems we have investigated (dimyristoylphosphatidyl ethanolamine bilayers and mixed systems composed by dimyristoylphosphatidyl ethanolamine/dimyristoylphosphatidylcholine mixtures and dimyristoylphosphatidyl ethanolamine/phosphatidic acid dipalmitoyl mixtures). The rate constants governing the reactions at the bilayer interfaces have been evaluated for K+ and Cl- ions, as a function of temperature, from 5 to 35 degrees C and bulk ionic concentrations from 0.02 to 0.2 M. Finally, a comparison between the original model of Skinner and the modified version is presented, and the advantages of this new formulation are briefly discussed.     

The interaction of hydrophobic ions with lipid bilayer membranes.

Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and L?uger (J. Membrane Biol. 5:225, 1971). This medel visualizes as three distinct steps the interfacial absorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions absorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon absorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon absorption, of a highly ordered water structure which surrounds the hydrophic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead by attributed to a more complex effect equivalent to a reduction of membrane fluidity.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.     

Spontaneous vesicle formation at lipid bilayer membranes.

Unilamellar vesicles are observed to form spontaneously at planar lipid bilayers agitated by exothermic chemical reactions. The membrane-binding reaction between biotin and streptavidin, two strong transmembrane neutralization reactions, and a weak neutralization reaction involving an "antacid" buffer, all lead to spontaneous vesicle formation. This formation is most dramatic when a viscosity differential exists between the two phases bounding the membrane, in which case vesicles appear exclusively in the more viscous phase. A hydrodynamic analysis explains the phenomenon in terms of a membrane flow driven by liberated reaction energy, leading to vesicle formation. These results suggest that energy liberated by intra- and extracellular chemical reactions near or at cell and internal organelle membranes can play an important role in vesicle formation, membrane agitation, or enhanced transmembrane mass transfer.     

Understanding peptide interactions with the lipid bilayer: a guide to membrane protein engineering

In recent years, studies of the interactions of designed hydrophobic and amphipathic polypeptides with biological membranes have progressed considerably. In-plane and transmembrane helical domains have been engineered as well as sequences that exhibit dynamic distributions of different topologies. These sequences not only help our understanding of the thermodynamic interaction contributions that determine peptide orientation, but also exhibit interesting biological functions as autonomous units. In addition, helices are considered to be folding intermediates of multi-spanning membrane proteins. The specificity and thermodynamic stability of transmembrane helix-helix interactions or the assembly of beta sheets at the membrane surface have, therefore, become a focus of ongoing research.     

Effect of peptide PV on the ionic permeability of lipid bilayer membranes

This paper reports the effects of peptide PV (primary structure: cyclo-(D-val-L-pro-L-val-D-pro)_δ) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (G_m) induced by PV in either Na⁺ or K⁺ medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in G_m in K⁺ medium is about 10⁴ times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K⁺ ≳ Rb⁺ > Cs⁺ > NH₄ ⁺ > TI⁺ > Na⁺ > Li⁺; the ratio of GGm in K⁺ to that in Na⁺ is about 10 for PV compared to > 10³ for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na⁺ or K⁺ salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10^–5 M. From a model which assumes that V_m equals the equilibrium potential for the PV-cation complexes (MS ⁺) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10^–5 cm s^–1, which is about 10^–7 times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of V_m are in agreement with the calculated equilibrium potential for MS⁺ over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS⁺ concentrations at the membrane surfaces.     

The interaction of detergents with bilayer lipid membranes

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025–0.15 mM; dodecyl sulphate 0.82 in the range 0.01–1 mM; sodium deoxycholate 1.03 in the range 0.01–1 mM and sodium cholate 1.37 in the range 0.1–10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.     

Stabilization of bilayer lipid membranes with polymeric resins

Bilayer lipid membranes on the bases of monoglyceride of oleic acid with the inclusion of polysterene are described. The inclusion of the polymer allows to obtain high stable membranes without sufficient changes in the nature of their conductivity.     

The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes

Gramicidin S (GS) is a cyclic decapeptide of primary structure [cyclo-(Val-Orn-Leu-D-Phe-Pro)(2)] secreted by Bacillus brevis. It is a powerful antimicrobial agent with potent cidal action on a wide variety of Gram-negative and Gram-positive bacteria as well as on several pathogenic fungi. Unfortunately, however, GS is rather non-specific in its actions and also exhibits a high hemolytic activity, limiting its use as an antibiotic to topical applications. In a wide variety of environments, the GS molecule exists as a very stable amphiphilic antiparallel beta-sheet structure with a polar and a non-polar surface. Moreover, the large number of structure-activity studies of GS analogs which have been carried out indicate that this 'sidedness' structure is required for its antimicrobial action. In this review, we summarize both published and unpublished biophysical studies of the interactions of GS with lipid bilayer model and with biological membranes. In general, these studies show that GS partitions strongly into liquid-crystalline lipid bilayers in both model and biological membranes, and seems to be located primarily in the glycerol backbone region below the polar headgroups and above the hydrocarbon chains. The presence of GS appears to perturb lipid packing in liquid-crystalline bilayers and GS can induce the formation of inverted cubic phases at lower temperatures in lipids capable of forming such phases at higher temperature in the absence of peptide. The presence of GS at lower concentrations also increases the permeability of model and biological membranes and at higher concentrations causes membrane destabilization. There is good evidence from studies of the interaction of GS with bacterial cells that the destruction of the integrity of the lipid bilayer of the inner membrane is the primary mode of the antimicrobial action of this peptide. The considerable lipid specificity of GS for binding to and destabilization of lipid bilayer model membranes indicates that the design of GS analogs with an improved antimicrobial potency and a markedly decreased toxicity for eukaryotic cell plasma membranes should be possible.     

Binding of polylysine to charged bilayer membranes: molecular organization of a lipid.peptide complex.

The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.