Peptide binding to lipid bilayers. Binding isotherms and zeta-potential of a cyclic somatostatin analogue

The binding of the cyclic somatostatin analogue SMS 201-995, (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, to neutral and negatively charged lipids was investigated with a centrifugation assay and with electrophoretic and monolayer methods. Monolayers and bilayers were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), either in pure form or in a 75/25 (mol/mol) mixture. The expansion of monolayer films demonstrated the intercalation of the peptide between the lipid molecules with a surface area requirement of 135 A2 per peptide molecule, indicating a parallel alignment of the peptide long axis with the membrane surface. Above a limiting pressure of 32.5 mN/m for POPC and 38.5 mN/m for POPG, peptide penetration was no longer possible. The peptide binding isotherm could be measured for mixed POPC/POPG bilayers up to a peptide concentration of 0.5 mM. Due to electrostatic attraction, binding between the positively charged peptide and the negatively charged membrane surface was enhanced as compared to the binding to a neutral membrane. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherm as well as the electrophoretic zeta-potential measurement could be described by the same partition equilibrium with a surface partition constant of Kp = 36 +/- 4 M-1 (at 0.1 M NaCl). About 60-70% of SMS 201-995 is probably embedded in the headgroup region with little penetration into the lipid core. The partition constant increases with increasing salt concentration or with decreasing lipid lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

Free-energy determinants of alpha-helix insertion into lipid bilayers.

A detailed treatment is provided of the various free-energy terms that contribute to the transfer of a polyalanine alpha-helix from the aqueous phase into lipid bilayers. In agreement with previous work, the hydrophobic effect is found to provide the major driving force for helix insertion. However, an opposing effect of comparable magnitude is also identified and is attributed to the large free-energy penalty associated with the desolvation of peptide hydrogen bonds on transfer to the low dielectric environment of the bilayer. Lipid perturbation effects as well as the entropy loss associated with helix immobilization in the bilayer are also evaluated. Two configurations of a membrane-bound 25mer polyalanine helix were found to be lower in free energy than the isolated helix in the aqueous phase. The first corresponds to the case of vertical insertion, in which a helix terminus protrudes from each side of the bilayer. The second minimum is for the case of horizontal insertion, for which the helix is adsorbed upon the surface of the bilayer. The calculated free-energy minima are found to be in good agreement with recent measurements of related systems. Large free-energy barriers resulting from desolvation of unsatisfied hydrogen-bonding groups at the helix termini are obtained for both insertion processes. The barriers for insertion are significantly reduced if the helix termini are assumed to be "capped" through the formation of hydrogen bonds with polar sidechains. For uncapped helices, our results support recently proposed models in which helices are inserted by first adsorbing on the membrane surface and then having one terminus "swing around" so as to penetrate the bilayer.     

NMR studies on phospholipid bilayers. Some factors affecting lipid distribution.

1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.     

Magnetic resonance studies on the binding of etomidate to lipid bilayers

Physicochemical studies on the binding of etomidate, a fast acting anaesthetic, with lipid bilayers have been carried out. ESR spin labeling studies indicate that the gel to liquid crystalline phase transition of dipalmitoyl phosphatidyl choline (DPPC) vesicles retains its cooperative nature on incorporation of the anaesthetic. For a 5:1 lipid to drug molar ratio, the phase transition occurs at an unusually lower temperature than those observed with other drug-DPPC systems. Results of 13C NMR and 1H NOE experiments suggest that the drug molecules reside in the close proximity of the terminal of hydrocarbon chains of the lipid molecules. 31P NMR and Electron Microscopic experiments indicate that the presence of etomidate alters the normal lamellar structure of DPPC vesicles into hexagonal (HII) type. Based on these observations, a model for drug-lipid binding has been proposed.     

Peptide binding to lipid bilayers. Nonclassical hydrophobic effect and membrane-induced pK shifts.

The binding of the cyclic peptide (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, a somatostatin analogue (SMS 201-995), and the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to lipid membranes was investigated with high-sensitivity titration calorimetry. The binding enthalpy of the peptide was found to vary dramatically with the vesicle size. For highly curved vesicles with a diameter of d congruent to 30 nm, the binding reaction was enthalpy-driven with delta H congruent to -7.0 +/- 0.3 kcal/mol; for large vesicles with more tightly packed lipids, the binding reaction became endothermic with delta H congruent to +1.0 +/- 0.3 kcal/mol and was entropy-driven. In contrast, the free energy of binding was almost independent of the vesicle size. The thermodynamic analysis suggests that the observed enthalpy-entropy compensation of about 8 kcal/mol can be related to a change in the internal tension of the bilayer and is brought about by an entropy increase of the lipid matrix. The "entropy potential" of the membrane may have its molecular origin in the excitation of the hydrocarbon chains to a more disordered configuration and may play a more important role in membrane partition equilibria than the classical hydrophobic effect. The binding of the peptide to the membrane surface induced a pK shift of the peptide terminal amino group. Neutral membranes were found to destabilize the NH3+ group, leading to a decrease in pK; negatively charged membranes, generated an apparent increase in pK due to the increase in proton concentration near the membrane surface. No pK shifts were seen for TNS. Titration calorimetry combined with the Gouy-Chapman theory can be used to determine both the reaction enthalpy and the binding constant of the membrane-binding equilibrium.     

Reversible adsorption and nonreversible insertion of Escherichia coli alpha-hemolysin into lipid bilayers.

Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.     

Effect of changing the size of lipid headgroup on peptide insertion into membranes.

Adsorption of amphiphilic peptides to the headgroup region of a lipid bilayer is a common mode of protein-membrane interactions. Previous studies have shown that adsorption causes membrane thinning. The degree of the thinning depends on the degree of the lateral expansion caused by the peptide adsorption. If this simple molecular mechanism is correct, the degree of lateral expansion and consequently the membrane thinning should depend on the size of the headgroup relative to the cross section of the hydrocarbon chains. Previously we have established the connection between the alamethicin insertion transition and the membrane thinning effect. In this paper we use oriented circular dichroism to study the effect of varying the size of the headgroup, while maintaining a constant cross section of the lipid chains, on the insertion transition. A simple quantitative prediction agrees very well with the experiment.     

The binding of organic ions to phospholipid bilayers

The binding of organic anions and cations, mainly tetraphenylboride and tetraphenylarsonium, to phospholipid membranes has been studied using an NMR method. Binding is appreciable and is affected by cholesterol in the membrane and counterions in solution. The passage of the organic anions through the membrane has also been followed. These measurements indicate that it is naive to use organic anions to measure membrane potentials in a simple manner.     

Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles

We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar vesicles (diameter approximately 100 nm) composed of phosphatidylcholine and phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large unilamellar vesicles are significantly larger than the peptide, the correlation times for the free and bound peptide could be distinguished using single color autocorrelation measurements. The molar partition coefficients calculated from the FCS measurements were comparable to those obtained from binding measurements of radioactively labeled MARCKS(151-175) using a centrifugation technique. Moreover, FCS can measure binding of peptides present at very low concentrations (1-10 nmolar), which is difficult or impossible with most other techniques. Our data indicate FCS can be an accurate and valuable tool for studying the interaction of peptides and proteins with lipid membranes.     

Catalyzed insertion of proteins into phospholipid membranes: specificity of the process

The process of insertion of intrinsic proteins into phospholipid membranes conjures up the thought of enormous energy barriers but is a routine occurrence in cells. Proteinaceous complexes responsible for protein targeting/translocation/insertion into membranes have been studied intensively. However, the mitochondrial voltage-dependent anion channel (VDAC), can insert into phospholipid membranes by an auto-catalytic process called "auto-directed insertion." This process results in an oriented insertion of VDAC channels and an increase in insertion rate per unit area of 10 orders of magnitude. Here we report that VDAC catalyzes the insertion of PorA/C1 and KcsA by increasing their calculated insertion rate per unit area by 9 orders of magnitude with no detectable effect on the insertion of alpha-hemolysin. This was measured as a reduction in the delay before the first insertion of these proteins. Gramicidin and PorA/C1 accelerate the calculated insertion rate per unit area of VDAC by 8 and 9 orders of magnitude, respectively. Only PorA/C1 increases the overall rate of VDAC insertion (50-fold) over the self-catalyzed rate. Our results indicate that catalyzed insertion of proteins into phospholipid membranes does not arise simply from disturbance of the phospholipid membrane because it shows strong specificity.     

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde concentration in the outer monolayer.     

Spectroscopic evidence for a preferential location of lidocaine inside phospholipid bilayers

We examined the effect of uncharged lidocaine on the structure and dynamics of egg phosphatidylcholine (EPC) membranes at pH 10.5 in order to assess the location of this local anesthetic in the bilayer. Changes in the organization of small unilamellar vesicles were monitored either by electron paramagnetic resonance (EPR)-in the spectra of doxyl derivatives of stearic acid methyl esters labeled at different positions in the acyl chain (5-, 7-, 12- and 16-MeSL)-or by fluorescence, with pyrene fatty-acid (4-, 6-, 10- and 16-Py) probes. The largest effects were observed with labels located at the upper positions of the fatty-acid acyl-chain. Dynamic information was obtained by 1H-NMR. Lidocaine protons presented shorter longitudinal relaxation times (T(1)) values due to their binding, and consequent immobilization to the membrane. In the presence of lidocaine the mobility of all glycerol protons of EPC decreased, while the choline protons revealed a higher degree of mobility, indicating a reduced participation in lipid-lipid interactions. Two-dimensional Nuclear Overhauser Effect experiments detected contacts between aromatic lidocaine protons and the phospholipid-choline methyl group. Fourier-transform infrared spectroscopy spectra revealed that lidocaine changes the access of water to the glycerol region of the bilayer. A "transient site" model for lidocaine preferential location in EPC bilayers is proposed. The model is based on the consideration that insertion of the bulky aromatic ring of the anesthetic into the glycerol backbone region causes a decrease in the mobility of that EPC region (T(1) data) and an increased mobility of the acyl chains (EPR and fluorescence data).     

Nonelectrostatic contributions to the binding of MARCKS-related protein to lipid bilayers.

The association of various protein constructs of MARCKS-related protein (MRP) lacking the myristoyl moiety or the basic effector domain (ED) or both to neutral and acidic supported planar phospholipid bilayer membranes has been monitored using two-mode optical waveguide spectroscopy. The importance of the myristoyl moiety for interaction with both neutral and acidic membranes is demonstrated but unmyristoylated MRP still binds appreciably to neutral membranes, albeit less than to acidic membranes. Only when both the myristoyl moiety and the ED are excised does the interaction fall to zero in the case of the acidic membranes, with very small residual binding still detectable in the presence of neutral membranes. These results point to the importance of hydrophobic interactions apart from those associated with the myristoyl moiety in the association of MRP with membranes. The ED is well endowed with hydrophobic as well as with basic residues, and the former are chiefly responsible for binding unmyristoylated MRP to neutral membranes: The very small residual attraction between MRP lacking both the myristoyl moiety and the ED is completely outweighed by electrostatic repulsion between the net acidic MRP and the acidic lipid head groups.     

Statistical thermodynamic analysis of peptide and protein insertion into lipid membranes.

A statistical thermodynamic approach is used to analyze the various contributions to the free energy change associated with the insertion of proteins and protein fragments into lipid bilayers. The partition coefficient that determines the equilibrium distribution of proteins between the membrane and the solution is expressed as the ratio between the partition functions of the protein in the two phases. It is shown that when all of the relevant degrees of freedom (i.e., those that change their character upon insertion into the membrane) can be treated classically, the partition coefficient is fully determined by the ratio of the configurational integrals and thus does not involve any mass-dependent factors, a conclusion that is also valid for related processes such as protein adsorption on a membrane surface or substrate binding to proteins. The partition coefficient, and hence the transfer free energy, depend only on the potential energy of the protein in the membrane. Expressing this potential as a sum of a "static" term, corresponding to the equilibrium (minimal free energy) configuration of the protein in the membrane, and a "dynamical" term representing fluctuations around the equilibrium configuration, we show that the static term contains the "solvation" and "lipid perturbation" contributions to the transfer free energy. The dynamical term is responsible for the "immobilization" free energy, reflecting the loss of translational and rotational entropy of the protein upon incorporation into the membrane. Based on a recent molecular theory of lipid-protein interactions, the lipid perturbation and immobilization contributions are then expressed in terms of the elastic deformation free energy resulting from the perturbation of the lipid environment by the foreign (protein) inclusion. The model is formulated for cylindrically shaped proteins, and numerical estimates are given for the insertion of an alpha-helical peptide into a lipid bilayer. The immobilization free energy is shown to be considerably smaller than in previous estimates of this quantity, and the origin of the difference is discussed in detail.     

Segregation of chlorophyll a incorporated into lipid bilayers.

Absorption and fluorescence spectra are reported for chlorophyll a incorporated into a number of aqueous phospholipid dispersions. Absorption spectra show that in dipalmitoylphosphatidylcholine bilayers, monomeric and oligomeric forms of chlorophyll a are present in both the gel and liquid crystalline phases. The formation of aggregates of chlorophyll a is reflected in the fluorescence spectra by a marked concentration quenching. In bilayers conatining small proportions of chlorophyll a, a marked increase in aggregation occurs at the transition temperatures that can be detected calorimetrically. At higher concentrations (greater than 1 chlorophyll:100 lipid), the "pretransition" is abolished in the phosphatidylcholines, and the main transition is broadened, consistent with an orientation for the chlorophyll a with the chlorine ring in the head group region and the phytol chain in the fatty acid chain region of the bilayer. In mixtures of saturated and unsaturated lipids, there is no preferential segregation of the chlorophyll a into the unsaturated lipid.     

Calcium-dependence of laminin binding to phospholipid membranes

Laminin is the most abundant noncollagenous protein in basement membranes. Its selfassembly has been studied in solution, and it has been established that calcium ions induce partially reversible aggregation. The behavior at a lipid membrane surface is of greater biological significance, but it is difficult to study quantitatively binding kinetics at a surface. The present work uses a powerful new integrated optics technique to measure the absorption and aggregation of the laminin–nidogen at a bilayer lipid membrane surface. It is found that the binding of a single layer of laminin at the lipid membrane is independent of the presence of calcium, but that the building up of multilayer laminin membranes requires calcium, and that these may not be destroyed by a calcium-complexing agent. © 1993 John Wiley & Sons, Inc.     

Anion binding to lipid bilayers: a study using fluorescent lipid probes

Anion-induced fluorescence quenching of lipid probes incorporated into the liposomal membrane was used to study the binding of anions to the lipid membrane. Lipid derivatives bearing nonpolar fluorophore located either in the proximity of the polar headgroups (anthrylvinyl-labelled phosphatidylcholine, ApPC; methyl 4-pyrenylbutyrate, MPB) or in the polar region (rhodamine 19 oleyl ester, OR19) of the bilayer were used as probes. The binding of iodide to the bilayers of different compositions was studied. Based on the anion-induced quenching of the fluorescence, the isotherm of adsorption of the quencher (iodide) to the membrane was plotted. For anions, which are non-quenchers or weak quenchers (thiocyanate, perchlorate or trichloroacetate), the binding parameters were obtained from the data of the competitive displacement of iodide by these anions. The association constants of the anion binding to the bilayer (Ka) were determined for the stoichiometry of 1 ion/1 lipid and also for the case of independent anion binding. At the physiological concentration of the salt, which does not bind noticeably to the membrane (150 mM NaCl), anion binding could be satisfactorily described by the Langmuir isotherm. The approach applied here offers new possibilities for the studies of ion-membrane interactions using fluorescent probes.