Study of the interaction of myoglobin with lipid bilayer membranes by potentiodynamic method

For modeling the interaction of myoglobin with mitochondrial membranes, the adsorption of different ligand forms, the physiologically active reduced MbO2 and inactive oxidized met-Mb, on one of the surfaces of artificial bilayer lipid membrane (BLM) was studied using potentiodynamic technique known as the "capacity minimization" method. As mitochondrial membranes are negatively charged, BLM from the negatively charged palmitoyl-2-oleil-phosphatidyl glycerol (POPG) and neutral soybean phosphatidylcholine (lecithin) were used. It is shown that both myoglobins strongly interact with BLM in the pH range 6-8. The dependence of the potential difference between cis-and trans-surfaces of the lipid membrane (deltaE, mV) on the protein concentration is characteristic for the Langmuir adsorption isotherm, and the saturation level (deltaEmax, mV) corresponds to monolayer of myoglobin. The protein adsorption is essentially electrostatic in nature, as adsorption activity increases sharply in the case of the membrane from POPG: in a approximately 15-fold in the case of MbO2 and in a approximately 2.5 times for the met-Mb. The parameters of the MbO2 and met-Mb adsorption on BLM from lecithin and POPG do not change in the pH 6-8 range. It can be assumed that the anionic groups of phospholipids associate with the cationic groups of the protein, the charge state of those does not change in the pH 6-8 range. The most likely candidates for interaction with phospholipids of BLM are invariant lysines and arginines in the environment of the myoglobin heme cavity.     

Study of the interaction of myoglobin with lipid bilayer membranes by potentiodynamic method

For modeling the interaction of myoglobin with mitochondrial membranes, the adsorption of different ligand forms, the physiologically active reduced MbO₂ and inactive oxidized met-Mb, on one of the surfaces of artificial bilayer lipid membrane (BLM) was studied using a potentiodynamic technique known as the “capacity minimization” method. As mitochondrial membranes are negatively charged, BLM of the negatively charged palmitoyl-2-oleyl-phosphatidyl glycerol (POPG) and neutral soybean phosphatidylcholine (lecithin) were used. It is shown that both myoglobins strongly interact with BLM in the pH range 6–8. The dependence of the potential difference between cis-and trans-surfaces of the lipid membrane (ΔE, mV) on the protein concentration is characteristic of the Langmuir adsorption isotherm, and the saturation level (ΔE _max, mV) corresponds to monolayer of myoglobin. The protein adsorption is essentially electrostatic in nature, as adsorption activity increases sharply in the case of the membrane from POPG: ∼15-fold in the case of MbO₂ and ∼2.5 times for met-Mb. The parameters of the MbO₂ and met-Mb adsorption on BLM of lecithin and POPG do not change in the pH 6–8 range. It can be assumed that the anionic groups of phospholipids associate with the cationic groups of the protein, the charge state of those does not change in the pH 6–8 range. The most likely candidates for interaction with phospholipids of BLM are invariant lysines and arginines in the environment of the myoglobin heme cavity.     

Lipid and cell membranes in the presence of gadolinium and other ions with high affinity to lipids. 2. A dipole component of the boundary potential on membranes with different surface charge

When Gd3+, a trivalent lanthanide, binds phospholipids with a high affinity, it elicits strong electrostatic effects on the surface of the lipid bilayer. Two experimental methods were applied to monitor the changes in the boundary and surface potentials induced by Gd3+ adsorption on liposomes and planar lipid bilayer membranes (BLM) made from phosphatidylserine (PS), phosphatidylcholine (PC) and their mixtures. The membrane surface charge density was changed by either varying the PS/PC ratio or by changing the degree of PS headgroup ionization in the range of pH between 2.5 and 7.5. The Gouy-Chapman-Stern (GCS) theory combined with the condition of mass balance in the experimental cell was used for quantitative treatment of ion adsorption and related changes in the diffuse part of the electrical double layer (surface potential). Data obtained using microelectrophoresis of liposome suspensions were well described within the framework of the modified GCS theory with constants of 5.10(4) and 10(3) M-1 for Gd3+ association with PS and PC, respectively (Yu. A. Ermakov, A. Z. Averbakh, and S. I. Sukharev, Biol. Membrany 14:434-445 (1997) (in Russian)). The intramembrane field compensation (IFC) technique used to study Gd3+ adsorption on planar lipid bilayers by monitoring the entire boundary potential gave completely different results. An observed drastic difference (approximately 140 mV) between the changes of boundary and surface potential was interpreted as the change in the dipole potential induced by binding of Gd3+. The magnitude of the surface dipole increased with the concentration of PS in PS/PC mixtures and became significant at most negative surface charges (more than 80% of PS in the mixture) and strongly correlated with the degree of PS ionization at different pH. The nature of structural changes at the membrane/water interface induced by Gd(3+)-PS interaction and possible lipid clusterization are discussed in the context of their biological importance.     

Electrostatic effects upon adsorption and desorption of polylysines on the surface of lipid membranes of different composition

Electrokinetic measurements are carried out in suspensions of liposomes made from mixtures of charged (cardiolipin, CL) and neutral (phosphatidylcholine, PC) lipids in the presence of lysine and lysine-based polypeptides. Neither monolysine nor polylysines adsorbed on neutral (PC) membranes. In the case of negatively charged membranes (CL/PC) all polypeptides showed a sharp dependence of liposome electrophoretic mobility on the amount of polymer added to the cell. In suspension of cardiolipin liposomes the position of zero charge point coincided for all high-molecular polylysines; thus, pentalysine neutralizes the membrane surface, whereas polycations with a higher polymerization degree change a sign of the surface charge. Electrophoretic mobility of liposomes in plateau range depended on the molecular weight of polylysines and composition of liposomes; for large macromolecules the absolute value came close to its value for the initial liposomes. Adsorption of polycations on planar bilayer lipid membranes (BLM) resulted in alteration of the boundary potential measured by the method of intramembranous field compensation (IFC). The electrokinetic measurements and IFC method gave close results in the case of lysine monomers; their surface concentration could be fitted by an isotherm of the molecule distribution between the membrane surface and solution. Considerable differences of the surface and boundary potentials found in the case of pentalysine, correspond to changes in the dipole component of boundary potential induced by the adsorbed molecules. Using the IFC method, the kinetics of the adsorption process before saturation was studied. The adsorption of polylysines was markedly slower (more than hour) than that of pentalysine (tens of min) or monolysine (minutes). Washout experiments showed that adsorption of penta-and monolysine on planar BLM was reversible, while that of high-molecular polylysines was practically irreversible.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.     

The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential.

The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.     

The amyloid precursor protein interacts with neutral lipids.

The amyloid protein precursor (APP) was incorporated into liposomes or phospholipid monolayers. APP insertion into liposomes required neutral lipids, such as L-alpha-phosphatidylcholine, in the target membrane. It was prevented in vesicles containing L-alpha-phosphatidylserine. The insertion was enhanced in acidic solutions, suggesting that it is modulated by specific charge/charge interactions. Surface-active properties and behaviour of APP were characterized during insertion of the protein in monomolecular films of L-alpha-phosphatidylcholine, L-alpha-phosphatidylethanolamine or L-alpha-phosphatidylserine. The presence of the lipid film enhanced the rate of adsorption of the protein at the interface, and the increase in surface pressure was consistent with APP penetrating the lipid film. The adsorption of APP on the lipid monolayers displayed a significant head group dependency, suggesting that the changes in surface pressure produced by the protein were probably affected by electrostatic interactions with the lipid layers. Our results indicate that the penetration of the protein into the lipid monolayer is also influenced by the hydrophobic interactions between APP and the lipid. CD spectra showed that a large proportion of the alpha-helical secondary structure of APP remained preserved over the pH or ionic strength ranges used. Our findings suggest that APP/membrane interactions are mediated by the lipid composition and depend on both electrostatic and hydrophobic effects, and that the variations observed are not due to major secondary structural changes in APP. These observations may be related to the partitioning of APP into membrane microdomains.     

The effect of S-layer protein adsorption and crystallization on the collective motion of a planar lipid bilayer studied by dynamic light scattering.

A dedicated dynamic light scattering (DLS) setup was employed to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes (BLM), over a previously inaccessible spread of frequencies (relaxation times ranging from 10(-2) to 10(-6) s) and wavevectors (250 cm(-1) < q < 38,000 cm(-1)). For a BLM consisting of 1,2-dielaidoyl-sn-3-glycero-phosphocholine (DEPC) doped with two different proportions of the cationic lipid analog dioctadecyl-dimethylammonium bromide (DODAB) we observed an increase of the lateral tension of the membrane with the DODAB concentration. The experimentally determined dispersion behavior of the transverse shear mode was in excellent agreement with the theoretical predictions of a first-order hydrodynamic theory. The symmetric adsorption of the crystalline bacterial cell surface layer (S-layer) proteins from Bacillus coagulans E38-66 to a weakly cationic BLM (1.5 mol % DODAB) causes a drastic reduction of the membrane tension well beyond the previous DODAB-induced tension increase. The likely reason for this behavior is an increase of molecular order along the lipid chains by the protein and/or partial protein penetration into the lipid headgroup region. S-layer protein adsorption to a highly cationic BLM (14 mol % DODAB) shows after 7 h incubation time an even stronger decrease of the membrane tension by a factor of five, but additionally a significant increase of the (previously negligible) surface viscosity, again in excellent agreement with the hydrodynamic theory. Further incubation (24 h) shows a drastic increase of the membrane bending energy by three orders of magnitude as a result of a large-scale, two-dimensional recrystallization of the S-layer proteins at both sides of the BLM. The results demonstrate the potential of the method for the assessment of the different stages of protein adsorption and recrystallization at a membrane surface by measurements of the collective membrane modes and their analysis in terms of a hydrodynamic theory.     

Adsorption of gamma-aminobutyric acid to phosphatidylserine membranes.

The interaction of the negatively-charged phosphatidylserine (PS) and gamma-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10(-5)-10(-4) M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of 14C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.     

Charge transfer mediated by nigericin in black lipid membranes.

Nigericin, in the concentration range (10(-6) M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K+ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K+. This charged complex accounts for the uncoupling effect observed in intact mitochondria.     

Measurements of local pH changes near bilayer lipid membrane by means of a pH microelectrode and a protonophore-dependent membrane potential. Comparison of the methods.

Shifts of pH near the bilayer lipid membrane (BLM) were measured in the absence of pH difference between bulk solutions by two methods, i.e. pH microelectrode and membrane potential recordings in the presence of a protonophore. A quantitative agreement of the results of both methods was obtained. The kinetics of the generation of potential induced by the addition of ammonium chloride was accounted for by the time of the diffusion through the unstirred layers. The thickness of the unstirred BLM layers was determined in the experiment.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

How lipids and proteins interact in a membrane: a molecular approach

Membrane proteins in a biological membrane are surrounded by a shell or annulus of 'solvent' lipid molecules. These lipid molecules in general interact rather non-specifically with the protein molecules, although a few 'hot-spots' may be present on the protein where anionic lipids bind with high affinity. Because of the low structural specificity of most of the annular sites, the composition of the lipid annulus will be rather similar to the bulk lipid composition of the membrane. The structures of the solvent lipid molecules are important in determining the conformational state of a membrane protein, and hence its activity, through charge and hydrogen bonding interactions between the lipid headgroups and residues in the protein, and through hydrophobic matching between the protein and the surrounding lipid bilayer. Evidence is also accumulating for the presence of 'co-factor' lipid molecules binding with high specificity to membrane proteins, often between transmembrane alpha-helices, and often being essential for activity.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Use of protein charge ladders to study electrostatic interactions during protein ultrafiltration

Recent studies have demonstrated the importance of electrostatic interactions in membrane systems, but there is still controversy about the underlying phenomena. Protein charge ladders, consisting of a set of chemical derivatives of a given protein that differ by single charge groups, were used to quantify the electrostatic interactions during protein ultrafiltration. Myoglobin charge ladders were generated by acylation, with the different derivatives analyzed simultaneously by capillary electrophoresis. Filtration experiments were performed using polyethersulfone and composite regenerated cellulose membranes, with the membrane charge determined from the streaming potential. As expected, the rejection increased as the protein became more heavily charged due to the increase in electrostatic repulsion. However, the transmission of the weakly charged myoglobin species increased dramatically at very low ionic strength. This increase in transmission was attributed to a shift in pH within the pore caused by hydrogen ion partitioning into the charged membrane. The sieving data were in good agreement with theoretical calculations accounting for the effects of this pH shift on the electrostatic interactions.     

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.     

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.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.