Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.     

Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.     

Theoretical calculation of the dielectric constant of a bilayer membrane.

The dielectric constant (epsilon) and refractive index (n) of a bilayer lipid membrane is determined from the known values of the polarizabilities of the carbon-carbon and carbon-hydrogen bonds. It is assumed that the hydrocarbon chains are hexagonally arranged in an all-trans conformation perpendicular to the plane of the membrane. The only variable in the calculation is the average separation between the chains and the theory relates epsilon to this separation. The calculation and results differ significantly from those presented in a 1968 publication by Ohki. It is shown that a thin membrane is not homogeneously polarized by the applied field. This effect is analysed and the dependence of epsilon on the membrane thickness is determined. The theoretical results are in good quantitative agreement with experimental measurements on bulk paraffins and on oriented multilayers of saturated fatty acids. The most important conclusion is that the dielectric constant for an applied field perpendicular to the membrane (which is the appropriate value for capacitance measurements) differs by only a few percent from the value for the macroscopic (bulk) liquid hydrocarbon. Thus the dielectric constant of a bilayer membrane can be approximated by the value for the appropriate bulk hydrocarbon.     

The molecular mechanism of action of the proton ionophore FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone).

We propose a simple model that accounts for the ability of the weak acid FCCP (Carbonylcyanide-p-trifluoromethoxyphenylhydrazone) to both transport protons across phospholipid bilayer membranes and uncouple oxidation from phosphorylation in mitochondria. Four parameters are required to characterize this model: the rate constant for the movement of A- across the membrane, kA, the rate constant for the movement of HA across the membrane, kHA, the adsorption coefficient of A- onto the membrane-solution interface, beta A, and the surface pK. These four parameters were determined from kinetic measurements on planar bilayer membranes using the charge-pulse and voltage-clamp techniques. We confirmed the adequacy of the model by determining each of these parameters independently, utilizing equilibrium dialysis, zeta potential, membrane potential, spectrophotometric, and conductance measurements. For a phosphatidylethanolamine bilayer the values of the parameters are kHA = 10(4)S-1, beta A = 3 10(-3) cm, and 6.0 less than pK less than 6.4. As predicted theoretically, the value of KA depends on both the applied voltage, V, and dielectric constant of the membrane, epsilon r; when V approaches zero and the membrane contains chlorodecane (epsilon r congruent to 2.7) kA = 700 s-1. If oxidation is coupled to phosphorylation by means of a delta microH+, and V er congruent to 2.7 for the inner membrane of the mitochondrion, the model predicts that FCCP should exert maximal uncoupling activity at a pH congruent to pK. This prediction agrees with the published experimental results.     

Photogating of ionic currents across lipid bilayers. Electrostatics of ions and dipoles inside the membrane.

The conductances of the lipophilic ions tetraphenylboride and tetraphenylphosphonium across a lipid bilayer can be increased or decreased, i.e., gated, by the photoformation of closed-shell metalloporphyrin cations within the bilayer. The gating can be effected by pulsed or continuous light or by chemical oxidants. At high concentrations of lipophilic anions where the dark conductance is saturated due to space charge in the bilayer, the photogated conductance can increase 15-fold. The formation of porphyrin cations allows the conductance to increase to its nonspace charge limited value. Conversely, the decrease of conductance in the light of phosphonium cations diminishes toward zero as the dark conductance becomes space charge limited. We present electrostatic models of the space charge limited conductance that accurately fit the data. One model includes an exponentially varying dielectric constant for the polar regions of the bilayer that allows an analytical solution to the electrostatic problem. The exponential variation of the dielectric constant effectively screens the potential and implies that the inside and outside of real dielectric interfaces can be electrically isolated from one another. The charge density, the distance into the membrane of the ions, about one-quarter of its thickness, and the dielectric constant at that position are determined by these models. These calculations indicate that there is insufficient porphyrin charge density to cancel the boride ion space charge and the following article proposes a novel ion chain mechanism to explain these effects. These models indicate that the positive potential arising from oriented carbonyl ester groups, previously used to explain the 10(3)-fold larger conductance of hydrophobic anions over cations, is smaller than previously estimated. However, the synergistic movement of the positive choline group into the membrane can account for the large positive potential.     

Optical and electrical properties of thin monoolein lipid bilayers

Summary Monoolein lipid bilayers were formed using a monolayer transfer technique and from dispersions of monoolein in squalene, triolein, 1-chlorodecane and 1-bromodecane. Measurements of optical reflectance and electrical capacitance were used to determine the thickness and dielectric constant of the bilayers. The thickness of the hydrocarbon region of the five bilayer systems ranged from 2.5 to 3.0 nm. Two of the bilayer systems (made from 1-chlorodecane and 1-bromodecane solvents) had a high dielectric constant (2.8 to 2.9) whereas the other bilayer systems had dielectric constants close to that of pure hydrocarbons (2.2). The charge-pulse technique was used to study the transport kinetics of three lipophilic ions and two ion carrier complexes in the bilayers. For the low dielectric constant bilayers, the transport of the lipophilic ions tetraphenylborate, tetraphenylarsonium and dipicrylamine was governed mainly by the thickness of the hydrocarbon region of the bilayer whereas the transport of the ion-carrier complexes proline valinomycin-K⁺ and valinomycin-Rb⁺ was nearly independent of thickness. This is consistent with previous studies on thicker monoolein bilayers. The transport of lipophilic anions across bilayers with a high dielectric constant was 20 to 50 times greater than expected on the basis of thickness alone. This agrees qualitatively with predictions based on Born charging energy calculations. High dielectric constant bilayers were three times more permeable to the proline valinomycin-K⁺ complex than were low dielectric constant bilayers but were just as permeable as low dielectric constant bilayers to the valinomycin-Rb⁺ complex.     

Lipid-water interface mediates reversible ionophore conformational change

A new procedure of conformational analysis was used to demonstrate that the ionophore conformation is mediated by its membrane environment. In the hydrophobic lipid matrix, the ionomycin-Ca++ complex adopts a conformation well suited for translocation across the interior of the membrane whereas at the lipid-water interface, the Ca++ ion is immersed into the aqueous phase in a position favorable to its complexation or decomplexation. The translocation of Ca++ across the lipid bilayer supposes a reversible transformation of the two conformers. The conformational analysis shows how the dielectric constant discontinuity existing at the lipid-water interface mediates the reversible transformation of one structure into the other.     

Proton transport through membranes induced by weak acids: A study of two substituted benzimidazoles

Summary We report here a kinetic study of the mechanism by which the weak acid TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) transports protons across phospholipid bilayer membranes. A previous kinetic study of the homologous dichloro compound, DTFB, revealed that the rate limiting step for proton translocation was the back diffusion of the neutral, HA, form of the weak acid; we conclude here that this is also the rate limiting step for proton translocation with TTFB. At high concentrations of either DTFB or TTFB the charged permeant species is an HA ₂ ^– complex. The kinetic analysis and independent measurements reveal that the permeability of the membrane to HA and adsorption coefficients of A^– and HA are an order of magnitude higher for TTFB than for DTFB. When either DTFB or TTFB was present in a solution where the pH was less than the pK of the weak acid, an unusual relaxation in the current was noted on application of a voltage step. The amplitude of the relaxation decreased as the voltage was increased. This relaxation is possibly due to a reorientation of the benzimidazole molecules at the membrane-solution interface. We also report experiments performed with DTFB on mitochondria. It was possible to reconcile these results with the bilayer data and, therefore, with the chemiosmotic hypothesis by postulating that the dielectric constant of the mitochondrial membrane is greater than that of a bilayer formed with decane as a solvent. To demonstrate the effect of dielectric constant on permeability, we replaced decane by 1-chlorodecane. This increased the capacitance of the artificial bilayer by a factor of two and the permeability of the bilayer to the A^– form of DTFB by two orders of magnitude.     

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.     

Alcohol effects on lipid bilayer permeability to protons and potassium: relation to the action of general anesthetics

Past work has shown that general anesthetics perturb the membranes of isolated synaptic vesicles, thereby increasing permeability to protons and inhibiting the ability of the vesicles to take up catecholamines. It has been proposed that such effects may produce anesthesia through inhibition of synaptic transmission. The mechanisms of perturbation is unknown. Two possible explanations include alterations of dielectric constant or production of defects as anesthetics partition into the bilayer phase. In order to choose between these alternatives, we measured the effect of nine alcohols and two alkanes on liposome permeability to protons and potassium. Ionic permeability was increased by alcohols and alkanes to similar degrees, thereby ruling out direct effects on the membrane dielectric constant caused by partitioning of anesthetics into the bilayer. Other experiments confirmed earlier reports that the enhanced permeability caused by anesthetics is not specific for protons. We conclude that these membrane perturbants act by increasing the number of transient, ion-conducting defects normally present in the bilayer structure.     

Local dielectric properties around polar region of lipid bilayer membranes

Summary Local dielectric constant was evaluated from the Stokes shifts of fluorescence spectra ofl--dansylphosphatidylethanolamine (DPE) incorporated into liposomes made of synthetic phosphatidylcholine (dipalmitoyl or distearoyl) or bovine brain phosphatidylserine. The evaluation was established as follows. First, the Stokes shift of DPE was assured to follow Mataga-Lippert's equation and was a function of the dielectric constant and the refractive index in some standard organic solvents. Second, the change of the refractive index did not contribute much to the change in the Stokes shift. Third, the time resolved fluorescence depolarization of DPE in liposomes showed that the cone wobbling diffusion was rapid relative to the fluorescence lifetime and therefore that the dielectric relaxation did not affect the evaluation of the constant in the polar region of membranes. We then investigated the characteristics of the local dielectric constant in the polar region of the lipid bilayer and found that the dielectric constant varies between 4 and 34 depending upon calcium binding and also gel/liquid-crystal phase transition. Such large changes of the local dielectric constant were further correlated with the dynamic structure of lipid bilayer membranes measured by conventional fluorescence depolarization techniques. The large changes of dielectric constant around the polar region suggest that electrostatic interactions at this region can be altered 10-fold by such ionic or thermotropic factors and therefore that local dielectric properties can play crucial roles in membrane functions.     

Supported phospholipid/alkanethiol biomimetic membranes: insulating properties.

A novel model lipid bilayer membrane is prepared by the addition of phospholipid vesicles to alkanethiol monolayers on gold. This supported hybrid bilayer membrane is rugged, easily and reproducibly prepared in the absence of organic solvent, and is stable for very long periods of time. We have characterized the insulating characteristics of this membrane by examining the rate of electron transfer and by impedance spectroscopy. Supported hybrid bilayers formed from phospholipids and alkanethiols are pinhole-free and demonstrate measured values of conductivity and resistivity which are within an order of magnitude of that reported for black lipid membranes. Capacitance values suggest a dielectric constant of 2.7 for phospholipid membranes in the absence of organic solvent. The protein toxin, melittin, destroys the insulating capability of the phospholipid layer without significantly altering the bilayer structure. This model membrane will allow the assessment of the effect of lipid membrane perturbants on the insulating properties of natural lipid membranes.     

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.     

Structure and location of amiodarone in a membrane bilayer as determined by molecular mechanics and quantitative x-ray diffraction.

Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.     

The redox properties of cytochromes b imposed by the membrane electrostatic environment.

The effect of the dipole potential field of extended membrane spanning alpha-helices on the redox potentials of b cytochromes in energy transducing membranes has been calculated in the context of a three phase model for the membrane. In this model, the membrane contains three dielectric layers; (i) a 40-A hydrophobic membrane bilayer, with dielectric constant em = 3-4, (ii) 10-20-A interfacial layers of intermediate polarity, ein = 12-20, that consist of lipid polar head groups and peripheral protein segments, and (iii) an external infinite water medium, ew = 80. The unusually positive midpoint potential, Em = +0.4 V, of the "high potential" cytochrome b-559 of oxygenic photosynthetic membranes, a previously enigmatic property of this cytochrome, can be explained by (i) the position of the heme in the positive dipole potential region near the NH2 termini of the two parallel helices that provide its histidine ligands, and (ii) the loss of solvation energy of the heme ion due to the low dielectric constant of its surroundings, leading to an estimate of +0.31 to +0.37 V for the cytochrome Em. The known tendency of this cytochrome to undergo a large -delta Em shift upon exposure of thylakoid membranes to proteases or damaging treatments is explained by disruption of the intermediate polarity (ein) surface dielectric layer and the resulting contact of the heme with the external water medium. Application of this model to the two hemes (bn and bp) of cytochrome b of the cytochrome bc1 complex, with the two hemes placed symmetrically in the low dielectric (em) membrane bilayer, results in Em values of hemes bn and bp that are, respectively, somewhat too negative (approximately -0.1 V), and much too positive (approximately +0.3 V), leading to a potential difference, Em(bp) - Em(bn), with the wrong sign and magnitude, +0.25 V instead of -0.10 to -0.15 V. The heme potentials can only be approximately reconciled with experiment, if it is assumed that the two hemes are in different dielectric environments, with that of heme bp being more polar.     

Continuum solvent model studies of the interactions of an anticonvulsant drug with a lipid bilayer

Valproic acid (VPA) is a short, branched fatty acid with broad-spectrum anticonvulsant activity. It has been suggested that VPA acts directly on the plasma membrane. We calculated the free energy of interaction of VPA with a model lipid bilayer using simulated annealing and the continuum solvent model. Our calculations indicate that VPA is likely to partition into the bilayer both in its neutral and charged forms, as expected from such an amphipathic molecule. The calculations also show that VPA may migrate (flip-flop) across the membrane; according to our (theoretical) study, the most likely flip-flop path at neutral pH involves protonation of VPA pending its insertion into the lipid bilayer and deprotonation upon departure from the other side of the bilayer. Recently, the flip-flop of long fatty acids across lipid bilayers was studied using fluorescence and NMR spectroscopies. However, the measured value of the flip-flop rate appears to depend on the method used in these studies. Our calculated value of the flip-flop rate constant, 20/s, agrees with some of these studies. The limitations of the model and the implications of the study for VPA and other fatty acids are discussed.     

Electrostatic interactions across a charged lipid bilayer

We present theoretical work in which the degree of electrostatic coupling across a charged lipid bilayer in aqueous solution is analyzed on the basis of nonlinear Poisson–Boltzmann theory. In particular, we consider the electrostatic interaction of a single, large macroion with the two apposed leaflets of an oppositely charged lipid bilayer where the macroion is allowed to optimize its distance to the membrane. Three regimes are identified: a weak and a high macroion charge regime, separated by a regime of close macroion–membrane contact for intermediate charge densities. The corresponding free energies are used to estimate the degree of electrostatic coupling in a lamellar cationic lipid–DNA complex. That is, we calculate to what extent the one-dimensional DNA arrays in a sandwich-like lipoplex interact across the cationic membranes. We find that, in spite of the low dielectric constant inside a lipid membranes, there can be a significant electrostatic contribution to the experimentally observed cross-bilayer orientational ordering of the DNA arrays. Our approximate analytical model is complemented and supported by numerical calculations of the electrostatic potentials and free energies of the lamellar lipoplex geometry. To this end, we solve the nonlinear Poisson–Boltzmann equation within a unit cell of the lamellar lipoplex using a new lattice Boltzmann method. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Soluble amyloid beta-oligomers affect dielectric membrane properties by bilayer insertion and domain formation: implications for cell toxicity

It is well established that Alzheimer's amyloid beta-peptides reduce the membrane barrier to ion transport. The prevailing model ascribes the resulting interference with ion homeostasis to the formation of peptide pores across the bilayer. In this work, we examine the interaction of soluble prefibrillar amyloid beta (Abeta(1-42))-oligomers with bilayer models, observing also dramatic increases in ion current at micromolar peptide concentrations. We demonstrate that the Abeta-induced ion conductances across free-standing membranes and across substrate-supported "tethered" bilayers are quantitatively similar and depend on membrane composition. However, characteristic signatures of the molecular transport mechanism were distinctly different from ion transfer through water-filled pores, as shown by a quantitative comparison of the membrane response to Abeta-oligomers and to the bacterial toxin alpha-hemolysin. Neutron reflection from tethered membranes showed that Abeta-oligomers insert into the bilayer, affecting both membrane leaflets. By measuring the capacitance of peptide-free membranes, as well as their geometrical thicknesses, the dielectric constants in the aliphatic cores of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers were determined to be epsilon = 2.8 and 2.2, respectively. The magnitude of the Abeta-induced increase in epsilon indicates that Abeta-oligomers affect membranes by inducing lateral heterogeneity in the bilayers, but an increase in the water content of the bilayers was not observed. The activation energy for Abeta-induced ion transport across the membrane is at least three times higher than that measured for membranes reconstituted with alpha-hemolysin pores, E(a) = 36.8 vs. 9.9 kJ/mol, indicating that the molecular mechanisms underlying both transport processes are fundamentally different. The Abeta-induced membrane conductance shows a nonlinear dependence on the peptide concentration in the membrane. Moreover, E(a) depends on peptide concentration. These observations suggest that cooperativity and/or conformational changes of the Abeta-oligomer particles upon transfer from the aqueous to the hydrocarbon environment play a prominent role in the interaction of the peptide with the membrane. A model in which Abeta-oligomers insert into the hydrophobic core of the membrane-where they lead to a local increase in epsilon and a concomitant reduction of the membrane barrier-describes the experimental data quantitatively.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

Calculations suggest a pathway for the transverse diffusion of a hydrophobic peptide across a lipid bilayer

Alamethicin is a hydrophobic antibiotic peptide 20 amino acids in length. It is predominantly helical and partitions into lipid bilayers mostly in transmembrane orientations. The rate of the peptide transverse diffusion (flip-flop) in palmitoyl-oleyl-phosphatidylcholine vesicles has been measured recently and the results suggest that it involves an energy barrier, presumably due to the free energy of transfer of the peptide termini across the bilayer. We used continuum-solvent model calculations, the known x-ray crystal structure of alamethicin and a simplified representation of the lipid bilayer as a slab of low dielectric constant to calculate the flip-flop rate. We assumed that the lipids adjust rapidly to each configuration of alamethicin in the bilayer because their motions are significantly faster than the average peptide flip-flop time. Thus, we considered the process as a sequence of discrete peptide-membrane configurations, representing critical steps in the diffusion, and estimated the transmembrane flip-flop rate from the calculated free energy of the system in each configuration. Our calculations indicate that the simplest possible pathway, i.e., the rotation of the helix around the bilayer midplane, involving the simultaneous burial of the two termini in the membrane, is energetically unfavorable. The most plausible alternative is a two-step process, comprised of a rotation of alamethicin around its C-terminus residue from the initial transmembrane orientation to a surface orientation, followed by a rotation around the N-terminus residue from the surface to the final reversed transmembrane orientation. This process involves the burial of one terminus at a time and is much more likely than the rotation of the helix around the bilayer midplane. Our calculations give flip-flop rates of approximately 10(-7)/s for this pathway, in accord with the measured value of 1.7 x 10(-6)/s.