C1q binding to liposomes is surface charge dependent and is inhibited by peptides consisting of residues 14-26 of the human C1qA chain in a sequence independent manner

Complement activation by anionic liposomes proceeds by antibody-independent, C1q-initiated activation of the classical pathway. Purified C1q bound to anionic liposomes in an acidic lipid concentration-dependent manner. Saturation binding, but not the apparent association constant, was enhanced by increasing the cardiolipin content of the liposomes or decreasing either the pH or ionic strength of the reaction mixture. These observations indicate the involvement of electrostatic factors in the binding. A highly cationic region in the collagen-like domain of C1q comprised of residues 14-26 of the C1qA polypeptide chain was assessed for involvement in liposome binding. This region has previously been shown to mediate C1q binding to other immunoglobulin-independent activators of the classical pathway of complement. Peptides containing residues 14-26 of C1qA, denoted C1qA14-26, inhibited C1q binding to and complement activation by anionic liposomes. The inhibitory capacity of these cationic peptides had no sequence or conformation specificity. Rather, the amount of positive charge on the peptides was the determining factor. When present in excess, peptides with five cationic residues inhibited C1q binding and complement activation; however, C1q peptides with only two cationic residues did not. In addition to the C1qA14-26 region, other parts of C1q that contain cationic residues may also be involved in C1q binding to anionic liposomes.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of the fatty acid-binding protein ReP1-NCXSQ with lipid membranes. Influence of the membrane electric field on binding and orientation

The regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ) is a 15 kDa soluble, intracellular protein that regulates the activity of the Na⁺/Ca ²⁺ exchanger in the squid axon. It is a member of the cellular retinoic acid-binding proteins family and the fatty acid-binding proteins superfamily. It is composed of ten beta strands defining an inner cavity and a domain of two short alpha helix segments. In this work, we studied the binding and orientation of ReP1-NCXSQ in anionic and zwitterionic lipid membranes using molecular dynamics (MD) simulations. Binding to lipid membranes was also measured by filtration binding assay. ReP1-NCXSQ acquired an orientation in the anionic membranes with the positive end of the macrodipole pointing to the lipid membrane. Potential of mean force calculations, in agreement with experimental measurements, showed that the binding to the anionic interfaces in low ionic strength was stronger than the binding to anionic interfaces in high ionic strength or to zwitterionic membranes. The results of MD showed that the electrostatic binding can be mediated not only by defined patches or domains of basic residues but also by a global asymmetric distribution of charges. A combination of dipole–electric field interaction and local interactions determined the orientation of ReP1-NCXSQ in the interface.     

Cooperative lipid-protein interaction. Effect of pH and ionic strength on polymyxin binding to phosphatidic acid membranes.

The binding of polymyxin-B to charged dipalmitoyl phosphatidic acid membranes has been studied as function of the external pH and of the ionic strength of the buffer solution. The phase transition curves were obtained by measuring the fluorescence depolarization of diphenyl hexatriene incorporated into the membrane with temperature. The molecular process of polymyxin binding was elucidated: 1. At an ionic strength of I greater than or equal to 0.1 mol/l a three step phase transition curve is found. A high-temperature step corresponds to the non-bound lipid. A lowered phase transition concerns to protein-bound lipid domains. This again is splitted into two steps. An inner core of the domain is characterized by a lipid-protein complex which is stabilized through hydrophobic and electrostatic interactions between polymyxin and the charged lipid. This core is surrounded by an outer belt of only hydrophobically bound molecules. This part shows a lower phase transition temperature than the inner core. 2. The binding curves of polymyxin to phosphatidic acid membranes depend strongly on the ionic strength of the water phase. The cooperativity of the binding process increases with increasing ionic strength and reaches a constant value at I greater than 0.2 mol/l. The maximum fraction of bound lipid decreases with increasing ionic strength. 3. The pH of the water phase strongly influences the cooperative binding process. At pH 6 a loss of cooperativity is observed at low ionic strength. Increasing the ion concentration to I = 0.3 mol/l recuperates the cooperativity of the binding process. At pH 3.0 no cooperative binding is obtained even at high ionic strength.     

Interactions of anionic phospholipids and phosphatidylethanolamine with the potassium channel KcsA

Fluorescence quenching methods have been used to study interactions of anionic phospholipids with the potassium channel KcsA from Streptomyces lividans. Quenching of the Trp fluorescence of KcsA reconstituted into mixtures of dioleoylphosphatidylcholine (DOPC) and an anionic phospholipid with dibromostearoyl chains is more marked at low mole fractions of the brominated anionic phospholipid than is quenching in mixtures of dibromostearoylphosphatidylcholine and nonbrominated anionic lipid. The quenching data are consistent with two classes of binding site for lipid on KcsA, one set corresponding to annular binding sites around KcsA to which DOPC and two-chain anionic phospholipids bind with similar affinities, the other set (non-annular sites) corresponding to sites at which anionic phospholipids can bind but from which DOPC is either excluded or binds with very low affinity. The binding constant for tetraoleoylcardiolipin at the annular sites is significantly less than that for DOPC, being comparable to that for dioleoylphosphatidylethanolamine. Tetraoleoylcardiolipin binds with highest affinity to the non-annular sites, the affinity for dioleoylphosphatidylglycerol being the lowest. The affinity for dioleoylphosphatidylserine decreases at high ionic strength, suggesting that electrostatic interactions between the anionic phospholipid headgroup and positively charged residues on KcsA are important for binding at the non-annular site. The effect of ionic strength on the binding of phosphatidic acid is less marked than on phosphatidylserine. The value of the binding constant for the non-annular site depends on the extent of Trp fluorescence quenching following from binding at the non-annular site. It is suggested that the non-annular site to which binding is detected in the fluorescence quenching experiments corresponds to the binding site for phosphatidylglycerol detected at monomer-monomer interfaces in x-ray diffraction studies.     

Membrane binding of the colicin E1 channel: activity requires an electrostatic interaction of intermediate magnitude.

In vitro channel activity of the C-terminal colicin E1 channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, I = 0.12 M, pH 4.0), the free energy of binding was delta G approximately -9 kcal/mol, with nonelectrostatic and electrostatic components, delta Gnel approximately -5 kcal/mol and delta Gel approximately -4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.     

Interaction of beta2-glycoprotein 1 with phosphatidylserine-containing membranes: ligand-dependent conformational alterations initiate bivalent binding

Beta2-glycoprotein 1 (beta2GP1), a 50 kDa serum glycoprotein that binds anionic phospholipid-containing membranes, plays a regulatory role in physiology and pathology. The protein is a member of the short consensus repeat (SCR) superfamily containing four typical repeating domains and an aberrant fifth domain constructed into an SCR-like core at the C-terminus. To investigate the contribution of the individual domains to the binding of beta2GP1, a series of sequential domain-deleted recombinant protein fragments were generated and assessed for their interaction with PS-containing vesicles. Spectral analyses of lipid binding-dependent alterations in tryptophan emission spectra revealed that the (single) tryptophan residues of the individual domains underwent binding-dependent conformational alterations. Depending on the ionic strength, some domains moved from polar to nonpolar environments, while others moved from less polar to more polar environments. Analysis of a series of acrylamide quenching and resonance energy transfer experiments indicated that the binding of N-terminal domain 1 to PS membranes exists in two, ionic strength-dependent, conformations. At low ionic strengths, domain 1 bound to the vesicles and induced their precipitation and/or aggregation. At physiologic ionic strengths, domain 1 detached from the membrane surface while the remaining domains maintained their association with the membrane. Under these conditions, membrane-bound conformationally altered domain 1 projects away from the membrane surface, enabling it to interact with other proteins and/or cell surface ligands or receptors.     

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.     

The effect of phospholipid vesicles on the kinetics of reduction of cytochrome c.

The rate of reduction of cytochrome c by ascorbate and by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of ionic strength and of binding to phospholipid vesicles (liposomes). Binding of cytochrome c to liposomes, which occursat low ionic strength, decreases the rate of reduction by ascorbate by a factor of up to 100, which can be primarily explained on electrostatic grounds. In the absence of liposomes, kinetics of reduction by the neutral pteridine derivative showed no ionic strength dependence. Binding of cytochrome c to liposomes increased the rate of reduction by pteridine. An estimation of the binding constant of cytochrome c to liposomes at 0.06 M ionic strength, pH 7, is given.     

Role of anionic lipid in bacterial membranes

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.     

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter

Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Partly folded states of bovine carbonic anhydrase interact with zwitterionic and anionic lipid membranes

The acidic, partly folded states of bovine carbonic anhydrase II (BCAII) were used as an experimental system to study the interactions of partly denatured proteins with lipid membranes. The pH dependence of their interactions with palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) membranes was studied. A filtration binding assay shows that acidic partly folded states of BCAII bind to POPC membranes. Fluorescence emission spectra from Trp residues of the bound protein are slightly shifted to shorter wavelength and can be quenched by a water-soluble quencher of fluorescence, indicating that the binding occurs without deep penetration of Trp residues into the membrane. The content of beta-structures of the protein in solution, as revealed by FT-IR spectroscopy, decreases in the partly folded states and the binding to POPC membrane occurs without further changes of secondary structure. In the presence of 0.1 M NaCl, a partly folded state self-aggregates and does not bind to POPC membrane. At acidic pH, BCAII binds to POPG membranes both at high and low ionic strength. The binding to the anionic lipid occurs with protein self-aggregation within the lipid-protein complexes and with changes in the secondary structure; large blue shifts in the fluorescence emission spectra and the decrease in the exposure to water-soluble acrylamide quencher of Trp fluorescence strongly suggest that BCAII penetrates the hydrocarbon domain in the POPG-protein complexes.     

Interactions of complement proteins C1q and factor H with lipid A and Escherichia coli: further evidence that factor H regulates the classical complement pathway

Proteins of the complement system are known to interact with many charged substances. We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids. Factor H inhibited C1q binding to anionic phospholipids, suggesting a role for factor H in regulating activation of the complement classical pathway by anionic phospholipids. To extend this finding, we examined interactions of C1q and factor H with lipid A, a well-characterized activator of the classical pathway. We report that C1q and factor H both bind to immobilized lipid A, lipid A liposomes and intact Escherichia coli TG1. Factor H competes with C1q for binding to these targets. Furthermore, increasing the factor H: C1q molar ratio in serum diminished C4b fixation, indicating that factor H diminishes classical pathway activation. The recombinant forms of the C-terminal, globular heads of C1q A, B and C chains bound to lipid A and E. coli in a manner qualitatively similar to native C1q, confirming that C1q interacts with these targets via its globular head region. These observations reinforce our proposal that factor H has an additional complement regulatory role of down-regulating classical pathway activation in response to certain targets. This is distinct from its role as an alternative pathway downregulator. We suggest that under physiological conditions, factor H may serve as a downregulator of bacterially-driven inflammatory responses, thereby fine-tuning and balancing the inflammatory response in infections with Gram-negative bacteria.     

An ion switch regulates fusion of charged membranes

Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way.     

DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.     

Electrostatically mediated interactions between cationic lipid-DNA particles and an anionic surface.

In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lipids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes prepared with 1, 2-dioleoyl-i-glycero-3-phosphoethanolamine (DOPE) or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 50 mol%, change the inherent electrophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid-DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (+/-) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (+/-) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal observed at a 1:1 net LDP to bead charge ratio (+/-). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP binding, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobility changes of the anionic latex beads.     

Electrostatic interaction of a K+ channel RCK domain with charged membrane surfaces

In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains. To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general functional behavior and identifying purification conditions. Standard detergent solubilization of recombinantly expressed MjK2 required the addition of a high NaCl concentration to the extraction buffer for MjK2 solubilization. The cytoplasmic RCK domain was identified as the region of MjK2 responsible for the dependence of solubilization on high salt concentrations since expression of an MjK2 construct lacking the transmembrane domain, MjK2cd, also required high salt concentrations for extraction from Escherichia coli lipids, a necessary step in the purification of both MjK2 and MjK2cd. MjK2 expression was able to weakly recover growth of K(+) uptake deficient LB2003 cells at 10 mM KCl, suggesting that the channel can conduct K(+) but has a low open probability. Purified MjK2 reconstituted in liposomes generated only limited Rb(+) uptake, blocked by BaCl(2). MjK2cd exhibited direct binding to PC/PS lipid vesicles with a molar partition coefficient (K(1)) of approximately 10(3) M(-)(1), which decreased with both an increase in the salt concentration and a decrease in the anionic lipid ratio. Lipid blot assays revealed that MjK2cd binds most strongly to lipids of increasingly negative charge. These results support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.     

Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis

Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.