Dissolution mechanism of supported phospholipid bilayer in the presence of amphiphilic drug investigated by neutron reflectometry and quartz crystal microbalance with dissipation monitoring

The influence and interaction of the ionizable amphiphilic drug amitriptyline hydrochloride (AMT) on a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid bilayer supported on a silica surface have been investigated using a combination of neutron reflectometry and quartz crystal microbalance with dissipation monitoring. Adding AMT solutions with concentrations 3, 12, and 50 mM leaves the lipid bilayer mainly intact and we observe most of the AMT molecules attached to the head-group region of the outer bilayer leaflet. Virtually no AMT penetrates into the hydrophilic head-group region of the inner leaflet close to the silica surface. By adding 200 mM AMT solution, the lipid bilayer dissolved entirely, indicating a threshold concentration for the solubilization of the bilayer by AMT. The observed threshold concentration is consistent with the observation that various bilayer structures abruptly transform into mixed AMT-DOPC micelles beyond a certain AMT-DOPC composition. Based on our experimental observations, we suggest that the penetration of drug into the phospholipid bilayer, and subsequent solubilization of the membrane, follows a two-step mechanism with the outer leaflet being removed prior to the inner leaflet.     

Electro-osmosis at the surface of phospholipid bilayer membranes

The electro-osmotic velocity is the velocity of a fluid near an interface produced by an electric field parallel to a surface. The velocity adjacent to fixed phospholipid bilayer membranes was measured by observing the velocity of small vesicles suspended in the fluid. The charge densities of the bilayers ranged from 0 to 1 electronic charge per lipid and experiments were performed at temperatures above and below the transition temperature of the phospholipid bilayer in 1, 10 and 100 mM NaCl solutions. The Helmholtz-Smoluchowski equation correctly predicted the electro-osmotic velocity from the known value of zeta potential of the phospholipid bilayer.     

Impact of phospholipid bilayer saturation on amyloid-β protein aggregation intermediate growth: A quartz crystal microbalance analysis

Evidence that membrane-associated amyloid aggregate growth can impart membrane damage represents one possible mechanism for the neurodegeneration associated with deposited amyloid-β protein (Aβ) aggregates in the brains of Alzheimer’s disease (AD) patients. This potential pathogenic event necessitates an understanding of the impact that cellular membrane composition may have on Aβ aggregate growth. In the current study, a quartz crystal microbalance (QCM) was employed to examine the growth of Aβ_1-40 aggregation intermediates on supported phospholipid bilayers (SPBs) assembled at the crystal surface. These surface-specific measurements illustrate that zwitterionic SPBs selectively bind aggregated but not monomeric protein, and these bound aggregates are capable of supporting nonsaturable reversible growth via monomer addition. Growth-capable Aβ_1-40 aggregation intermediates more readily bind SPBs composed of phospholipids with a greater degree of carbon saturation. Furthermore, kinetic analysis afforded by the quantitative real-time QCM measurements reveals that SPBs with greater saturation also better support the growth of bound Aβ_1-40 aggregation intermediates as a result of the slower dissociation of bound monomer rather than more efficient recognition between aggregate and monomeric protein. These findings correlate with epidemiological and experimental evidence that links increased dietary intake of polyunsaturated fatty acids to a reduced risk of AD.     

The interaction of lanthanide and calcium salts with phospholipid bilayer vesicles: The validity of the nuclear magnetic resonance method for determination of vesicle bilayer phospholipid surface ratios

Contrary to a recent report (B. Sears et al., Biochemistry 15 (1976) 1635), it has been determined that the ratio of the number of phospholipids on the inner and outer surfaces of phospholipid bilayer vesicles can be accurately determined by NMR paramagnetic ion shift reagent studies of vesicles. It is concluded that the metal interacts with all of the phospholipid on the exposed bilayer surface. A ratio of outer phospholipid to inner surface phospholipid of 2.1 ± 0.1 is obtained regardless of the nucleus studies, position of the nucleus relative to the metal ion binding site, molar ratio of metal to phospholipid over three orders of magnitude, or location of the metal ion on the inside or outside of the vesicle. Additionally, P-31 NMR studies using LaCl₃ and CaCl₂ indicate that Ca²⁺ weakly interacts with egg PC vesicles and than the lanthanides are adequate substitutes for Ca²⁺ since neither metal is found to perturb measurably the average polar head group conformation.     

Insulation of the conduction pathway of muscle transverse tubule calcium channels from the surface charge of bilayer phospholipid

Functional calcium channels present in purified skeletal muscle transverse tubules were inserted into planar phospholipid bilayers composed of the neutral lipid phosphatidylethanolamine (PE), the negatively charged lipid phosphatidylserine (PS), and mixtures of both. The lengthening of the mean open time and stabilization of single channel fluctuations under constant holding potentials was accomplished by the use of the agonist Bay K8644. It was found that the barium current carried through the channel saturates as a function of the BaCl2 concentration at a maximum current of 0.6 pA (at a holding potential of 0 mV) and a half-saturation value of 40 mM. Under saturation, the slope conductance of the channel is 20 pS at voltages more negative than -50 mV and 13 pS at a holding potential of 0 mV. At barium concentrations above and below the half-saturation point, the open channel currents were independent of the bilayer mole fraction of PS from XPS = 0 (pure PE) to XPS = 1.0 (pure PS). It is shown that in the absence of barium, the calcium channel transports sodium or potassium ions (P Na/PK = 1.4) at saturating rates higher than those for barium alone. The sodium conductance in pure PE bilayers saturates as a function of NaCl concentration, following a curve that can be described as a rectangular hyperbola with a half-saturation value of 200 mM and a maximum conductance of 68 pS (slope conductance at a holding potential of 0 mV). In pure PS bilayers, the sodium conductance is about twice that measured in PE at concentrations below 100 mM NaCl. The maximum channel conductance at high ionic strength is unaffected by the lipid charge. This effect at low ionic strength was analyzed according to J. Bell and C. Miller (1984. Biophysical Journal. 45:279-287) and interpreted as if the conduction pathway of the calcium channel were separated from the bilayer lipid by approximately 20 A. This distance thereby effectively insulates the ion entry to the channel from the bulk of the bilayer lipid surface charge. Current vs. voltage curves measured in NaCl in pure PE and pure PS show that similarly small surface charge effects are present in both inward and outward currents. This suggests that the same conduction insulation is present at both ends of the calcium channel.     

An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida.     

Elongation kinetics of polyglutamine peptide fibrils: a quartz crystal microbalance with dissipation study

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.     

Interaction of small molecules with phospholipid bilayer membranes: permeability studies

The passage of a phospholipid through the gel to liquid crystal phase transition is associated with an increase in the motional freedom of its fatty acyl chains as measured by spectroscopic techniques and an essentially isothermal absorption of heat as measured by differential scanning calorimetry (DSC). In addition, bilayers formed from that phospholipid display a permeability maximum for both non-electrolytes and electrolytes in the temperature region of the phase transition. In this study the sodium (and in some cases glucose) permeabilities of liposomes composed of either dimyristoyl or dipalmitoyl phosphatidylcholine plus dicetylphosphate were measured in the presence of a group of benzene and adamantane derivatives known to increase fatty acyl chain motion below the lipid transition temperature (T_c) and in the case of the adamantanes to also lower the T_c as measured by DSC. None of these compounds change the temperature at which the permeability maximum occurs despite their lowering of the phospholipid T_c. That is, in the presence of these additives there is observed an apparent dissociation between the phase transition and the permeability maximum. It is proposed that the permeability maximum normally observed in the temperature region of the T_c is associated with the completion of the ‘melting’ process. Hence a compound could cause early ‘melting’ of the bilayer but not change its permeability properties if the temperature at which the ‘melting’ process neared completion was not changed.     

Lipid-protein interactions in biomembranes studied through the phospholipid specificity of D-beta-hydroxybutyrate dehydrogenase

Since the biological membranes are fundamental units in the living cells, the studies of lipid-protein interactions are crucial for the understanding of their structure, functions and properties. Beside hydrophobic interactions between fatty acids chain of phospholipids and intrinsic membrane proteins, the interactions between charged groups of the protein with the polar heads of phospholipids generally confer the specificity which may be absolute or preferential. This paper reports essential results obtained these last few years with D-beta-hydroxybutyrate dehydrogenase (BDH) from inner mitochondrial membrane, one of the most interesting and best documented examples of a lipid-requiring enzyme. This is a review of the molecular basis--knowledge and strategy of study--of the lipid specificity for membrane protein functions.     

Changes of the phospholipid bilayer structure under the adsorption of ferricytochrome c on its surface]

Changes in the mobility of phospholipid molecules in liposomes membranes under adsorption ferricytochrome c on its surface were studied by means of NMR and EPR spectroscopy. It is found that the interaction of cytochrome molecules with vesicles causes the broadening of 1H-NMR signals of hydrophobic as well as polar groups in cardiolipin and phosphatidylcholine in the presence of lauric or phosphatidic acid. This broadening of 1H-NMR signals in hydrophobic groups may be caused by decrease in the rate of lateral diffusion of phospholipid molecules. The changes in the correlation time of hydrophobic spin-proub in liposomes containing phosphatydiloholine and cardiolipin with the increase of ferricytochrome c concentration were also observed. These changes suggest that the formation of protein-phospholipid clusters results in the impair of the regular structure of phospholipid bilayer.     

Assembly of the endoplasmic reticulum phospholipid bilayer: the phosphatidylcholine transporter

Phospholipids are synthesized and concomitantly inserted on the cytoplasmic surface of the endoplasmic reticulum. Assembly of the phospholipid bilayer requires translocation to the lumenal monolayer. The hypothesis that rat liver microsomes contain a protein transporter, or "flippase," for phosphatidylcholine was tested by measuring the transport of sn-1,2,-dibutyroylphosphatidylcholine (diC4PC). This homolog retains the polar head group, the portion of the phospholipid unable to undergo spontaneous transmembrane movement in vesicles, and its water solubility permits application of standard transport methods. DiC4PC entered the lumenal compartment of microsomal vesicles. Transport was saturable and was dependent on time, amount of microsomes, and an intact permeability barrier. DiC4PC transport was inhibited by structural analogs (but not by sn-2,3-diC4PC) and by treatment of microsomes with proteases, N-ethylmaleimide, and trinitrobenzenesulfonic acid. These data suggest that the transmicrosomal movement of diC4PC is protein mediated. DiC4PC was not transported across PC vesicles or red cell membranes, where PC translocation is slow.     

Analysis for the molecular motion of phospholipid bilayer with picosecond fluorometry

The viscosity and the molecular motion of phospholipid molecule in biological and artificial phospholipid bilayers were studied using picosecond fluorescence depolarization method with rod-like fluorophore, DPH. From the relationship between the viscosity in the lipid bilayer and the free space of phospholipid acyl-chain, it is concluded that the viscosity is determined mainly by the range of wobbling motion of the acyl-chain. Motion of polar head group was also measured by the same method with a newly synthesized fluorescent phospholipid, dipalmitoyl-phosphatidyl-umbelliferone. The rate and the range in the motion of head group were faster and larger than those of acyl-chain and gave the viscosity of head group layer to be 0.03 poise, which was about one tenth of that of acyl-chain layer in the liquid crystalline phase. This fact indicates that the head group layer would not resist the lateral diffusion of molecules in membrane and that the lateral diffusion rate of molecules could be estimated from the viscosity in the acyl-chain layer.     

Stabilization of the bio-membrane by small molecules: interaction of trehalose with the phospholipid bilayer

Anhydrobiotic organisms undergo periods of acute dehydration during their life cycle. It is of interest to understand how the biomembrane remains intact through such stress. A disaccharide, trehalose, which is metabolised during anhydrobiosis is found to prevent disruption of model membrane systems. Molecular modelling techniques are used to investigate the possible mode of interaction of trehalose with a model monolayer. The objective is to maximise hydrogen bonding between the two systems. A phospholipid matrix consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) is chosen to represent the monolayer. The crystal structure of DMPC reveals that there are two distinct conformers designated as A and B. An expansion of the monolayer, coplanar with its surface, results in the trehalose molecule being accommodated in a pocket formed by four B conformers. One glucose ring of the sugar rests on the hydrophobic patch provided by the choline methyls of an A conformer. Five hydrogen bonds are formed involving the phosphate oxygens of three of the surrounding B conformers. The model will be discussed with reference to relevant experimental data on the interaction.     

Interaction of the disaccharide trehalose with a phospholipid bilayer: a molecular dynamics study

The disaccharide trehalose is well known for its bioprotective properties. Produced in large amounts during stress periods in the life of organisms able to survive potentially damaging conditions, trehalose plays its protective role by stabilizing biostructures such as proteins and lipid membranes. In this study, molecular dynamics simulations are used to investigate the interaction of trehalose with a phospholipid bilayer at atomistic resolution. Simulations of the bilayer in the absence and in the presence of trehalose at two different concentrations (1 or 2 molal) are carried out at 325 K and 475 K. The results show that trehalose is able to minimize the disruptive effect of the elevated temperature and stabilize the bilayer structure. At both temperature, trehalose is found to interact directly with the bilayer through hydrogen bonds. However, the water molecules at the bilayer surface are not completely replaced. At high temperature, the protective effect of trehalose is correlated with a significant increase in the number of trehalose-bilayer hydrogen bonds, predominantly through an increase in the number of trehalose molecules bridging three or more lipid molecules.     

Molecular-dynamic simulation of phospholipid bilayers: Ion distribution at the surface of neutral and charged bilayer in the liquid crystalline state

The electric field and ion distribution at the surface of neutral and charged lipid bilayers (BeCl₂ and dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylserine (DPPC/DPPS) + KCl) were studied with molecular dynamic (MD) methods. It is shown that the contributions of lipid molecules, water and ions to the electric potential compensate each other in the region of the diffuse double layer and decrease the potential value close to zero. It is also demonstrated that the ion distribution at the charged surface is determined not only by the electrostatic ion-medium interaction. The total energy of this interaction was compared with the potential of mean ion force. It was shown that cations and anions have a different effect on the state of water molecules at the surface. The order parameter of water in the system DPPC + BeCl₂ and the Clion distribution have the extremum at the distance of 10 α atoms of the phospholipid glycerol. This position was chosen as the “electrical” interface of the electrical double layer (EDL) for all lipid systems studied. The potential of mean force of counter ions in EDL allows us to obtain the value of potential at the lipid surface suitable for experimental test of the MD data. This surface potential and surface charge density was found from MD simulation different electrolyte concentrations and DPPS content of 20, 40 and 60% in the mixture with DPPC and was shown to be in a good agreement with the Gouy-Chapman-Stern model upon fitting parameters close to their experimental values.     

Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides

Permeation of the cell membrane leading to cell death is a mechanism used by a large number of membrane-lytic peptides. Some are linear, mostly helical, and others contain one or more disulfide bonds forming beta-sheet or both beta-sheet and alpha-helix structures. They are all soluble in solution but when they reach the target membrane, conformational changes occur which let them associate with and lyse the membrane. Some lytic peptides are not cell-selective and lyse different microorganisms and normal mammalian cells, while others are specific to either type of cells. Despite extensive studies, the mode of action of membrane-lytic peptides is not fully understood and the basis for their selectivity towards specific target cells is not known. Many studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipathic alpha-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a 'barrel-stave' mechanism; and (ii) membrane destruction/solubilization via a 'carpet' mechanism. This review, which is focused on the different stages of membrane permeation induced by representatives of amphipathic alpha-helical antimicrobial and cell non-selective lytic peptides distinguishes between the 'carpet' mechanism, which holds for antimicrobial peptides versus the 'barrel-stave' mechanism, which holds for cell non-selective lytic peptides.     

Ligand binding to cytochrome P450 3A4 in phospholipid bilayer nanodiscs: the effect of model membranes

The membrane-bound protein cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning the membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high density lipoprotein particles, whose stability, monodispersity, and consistency are ensured by their self-assembly. We explore the energetics of four ligands (6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), alpha-naphthoflavone (ANF), miconazole, and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the scaffold proteins of Nanodiscs; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 microm for TNS, 5.8 microm for ANF, 0.45 microm for miconazole, and 0.45 microm for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment; ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site.     

Intrinsic differences in the perturbing ability of alkanols in bilayer: Action of phospholipase A2 on the alkanol-modified phospholipid bilayer

Summary The kinetic parameters for the steady-state rate of hydrolysis of egg phosphatidylcholine in multilamellar vesicles by bee venom phospholipase A₂ are measured in the presence of 27 alkanols and several organic solvents. In general, small nonpolar solutes like enflurane, tetrahydrofuran, benzene, chloroform and diethylether do not promote the hydrolysis of multilamellar vesicles. The rate of hydrolysis shows a biphasic dependence upon the alkanol concentration for all higher (C₅–C₉) alcohols examined, i.e., an optimal rate of hydrolysis is observed at a characteristic concentration for each alcohol. The alkanol to lipid mole ratio (D/L ratio) in the bilayer at the peak activating concentration of an alkanol was computed from its bilayer/water partition coefficient. The branched chain alcohols induce peak activation of hydrolysis at lowerD/L ratios in the bilayer than the corresponding straight chain analogs. Similarly, the longer chainn-alkanols at peak activating concentration have a lowerD/L ratio than the corresponding lower alcohols. Both theK _m andV _m for phosphatidylcholine increase as a function of the chain length of the activating alcohol. These kinetic parameters also depend upon the position of the substituents on the activating alcohols. Both theD/L ratio andV _m for an alcohol are found to change with the cross-sectional area of the activating alcohol across its long axis: alcohols with a more asymmetric cross-section exhibit higherV _m and a lowerD/L ratio. Such correlations ofV _m andD/L ratio with the molecular parameters of the alkanols are interpreted to suggest that the accessibility of the substrate molecule in the bilayer to the phospholipase is modulated by the free space introduced by the alkanols in the bilayer.Effect of tetradecane derivatives and A₂C (a membrane fluidizing agent) on the phase transition characteristics of DPPC bilayers, and their susceptibility to phospholipase A₂ from bee venom and pig pancreas is also reported. These solutes cause a broadening of the transition profile and reduce the size of the cooperative unit and the enthalpy of transition. These effects depend upon the mole fraction of a solute in the bilayer; however, equal concentrations of these solutes do not induce equal response. Susceptibility of the modified bilayers to phospholipase A₂ depends not only upon the structure of the solute but also upon the source of the enzyme. The data show that the activity of the membrane-bound enzyme is modulated to different extents by different solutes, and the bilayer perturbing ability of these solutes may be related to the asymmetry of their cross-sectional area and to the free space introduced by the alkanols in a bilayer.     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.