Cholesterol/phospholipid interactions in hybrid bilayer membranes

The interactions between cholesterol and saturated phospholipids in hybrid bilayer membranes (HBMs) were investigated using the interface-sensitive technique of vibrational sum frequency spectroscopy (VSFS). The unique sensitivity of VSFS to order/disorder transitions of the lipid acyl chains was used to determine the main gel to liquid crystal phase transition temperature, Tm, for HBMs of binary cholesterol/phospholipid mixtures on octadecanethiolate self-assembled monolayers. The phase transition temperature and the breadth of the transition were shown to increase with cholesterol content, and the phase boundaries observed in the cholesterol/phospholipid HBMs were comparable to the published phase diagrams of binary cholesterol/phospholipid vesicles. A thermodynamic assessment of the cooperative units of the HBM phase transitions revealed the presence of <10 nm diameter domains that were independent of the cholesterol composition.     

Simulations of lipid vesicle rupture induced by an adjacent supported lipid bilayer patch

Using a simple phenomenological model of a lipid bilayer and a surface, simulations were performed to study the bilayer-induced vesicle rupture probability as a vesicle adsorbs adjacently to a bilayer patch already adsorbed on the surface. The vesicle rupture probability was studied as a function of temperature, vesicle size, and surface-bilayer interaction strength. From the simulation data, estimates of the apparent activation energy for bilayer-induced vesicle rupture were calculated, both for different vesicle sizes and for different surface-bilayer interaction strengths.     

Modification of a supported lipid bilayer by polyelectrolyte adsorption

Addition of a weak polyelectrolyte, poly(methacrylic acid) (PMA), to a supported phospholipid bilayer made from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) depresses the melting temperature and alters the morphology of the bilayer in the gel phase. Ellipsometry measurements show that PMA adsorption lowers the phase transition temperature by 2.4 degrees C. Atomic force microscopy (AFM) showed no visible contrast in the fluid phase (above the melting temperature) but a rich morphology in the gel phase. In the gel phase, adsorption leads to formation of significantly less mobile phospholipid islands and other defects. One consequence of this lower mobility is a decrease in the implied cooperativity number of the phase transition, N, when polymer is added. Additionally, AFM images of the gel-phase bilayer show a highly defected structure that anneals significantly more slowly than in the absence of adsorbed polymer. Tentatively, we suggest that PMA preferentially decorates island and defect edges of the DMPC bilayer.     

Simulations of lipid transfer between a supported lipid bilayer and adsorbing vesicles

Recent experiments demonstrate transfer of lipid molecules between a charged, supported lipid membrane (SLB) and vesicles of opposite charge when the latter adsorb on the SLB. A simple phenomenological bead model has been developed to simulate this process. Beads were defined to be of three types, ‘n’, ‘p’, and ‘0’, representing POPS (negatively charged), POEPC (positively charged), and POPC (neutral but zwitterionic) lipids, respectively. Phenomenological bead–bead interaction potentials and lipid transfer rate constants were used to account for the overall interaction and transfer kinetics. Using different bead mixtures in both the adsorbing vesicle and in the SLB (representing differently composed/charged vesicles and SLBs as in the reported experiments), we clarify under which circumstances a vesicle adsorbs to the SLB, and whether it, after lipid transfer and changed composition of the SLB and vesicle, desorbs back to the bulk again or not. With this model we can reproduce and provide a conceptual picture for the experimental findings.     

Incorporation of the HERG potassium channel in a mercury supported lipid bilayer

The HERG potassium channel was incorporated in a mercury-supported tethered bilayer lipid membrane (tBLM) obtained by anchoring a thiolipid monolayer to the mercury surface and by self-assembling a lipid monolayer on top of it from a lipid film spread on the surface of an electrolyte solution. HERG was then incorporated in this tBLM from its micellar solution in Triton X-100, thus avoiding the use of vesicles in the preparation of the tBLM and of proteoliposomes in channel incorporation. The HERG "inward" current following a repolarization step was obtained by subtracting the current recorded upon addition of the specific inhibitor WAY from that recorded prior to this addition. This current was compared with that reported in the literature by the patch-clamp technique.     

Double cushions preserve transmembrane protein mobility in supported bilayer systems

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.     

Biophysical aspects of agar-gel supported bilayer lipid nembranes: a new method for forming and studying planar bilayer lipid membranes

Conventional bilayer lipid membranes (BLMs), formed by either the painting method or the Langmuir-Blodgett technique, lack the desired stability. This paper presents a simple method for forming long-lived BLMs on agar-gel supports. The supported BLM reported has a greatly improved mechanical stability and also has desirable dynamic properties. These self-assembled BLMs are of significant interest, in view of their similarity of biological membranes, their molecular dimension and their spontaneous formation.     

Interaction of cytolytic toxin CytB with a supported lipid bilayer: study using an acoustic wave device

An acoustic technique was used to monitor the interaction of the pore-forming cytolytic toxin CytB with a positively charged supported lipid bilayer. The acoustic device, which is based on a waveguide geometry, is sensitive to changes in the mass of the supported bilayer. The specificity of the interaction, rate and extent of the association, reversibility and effect of previous depositions of toxin were investigated. The CytB was found to bind irreversibly to the lipids at all fractional coverages even when the protein-to-lipid ratio was high enough to imply that the protein was associating with the external surface of the bilayer. The CytB formed stable structures with the bilayer at high protein surface concentrations and did not appear to disrupt the bilayer in the manner of a detergent. The rate of association with the bilayer was found to be directly proportional to the solution concentration of CytB at higher concentrations but appeared to be low at a CytB solution concentration of 5 microg mL(-1), leading to relatively low amounts of CytB being associated with the bilayer.     

Simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption

Recent experimental investigations of the kinetics of vesicle adsorption in solution on SiO2 demonstrate a thermally activated transition from adsorbed intact vesicles to a supported lipid bilayer. Our Monte Carlo simulations clarify the mechanism of this process. The model employed is an extension of the model used earlier to describe vesicle adsorption at room temperature. Specifically, it includes limitations of the adsorption rate by vesicle diffusion in the solution, and adsorption- and lipid-membrane-induced rupture of arriving and already adsorbed vesicles. Vesicles and lipid molecules, formed after rupture of vesicles, are considered immobile. With these ingredients, the model is able to quantitatively reproduce the temperature-dependent adsorption kinetics, including a higher critical surface concentration of intact vesicles for lower temperatures, and the apparent activation energy for the vesicle-to-bilayer transition E(a) approximately 5 kcal/mol.     

Ion-channel sensing of ferricyanide anion based on a supported bilayer lipid membrane.

Ferricyanide anion has usually been used as a marker of ion-channel sensors. In this work we first found that ferricyanide, itself, can act as a stimulus to regulate the permeability of sBLM prepared from didodecyldimethylammonium bromide (a kind of synthetic lipid) on a GC electrode. We used cyclic voltammetry and a.c. impedance to investigate this phenomenon. The interaction between sBLM and ferricyanide concerns time. Furthermore, we developed a sensor for ferricyanide anion. The ion-channel sensor is highly sensitive. It can detect ferricyanide concentration as low as 5 microM.     

Nitric oxide enhances the capacitance of self-assembled,supported bilayer lipid membranes

The effect of nitric oxide (NO) at biologically relevant concentrations on the electrochemical features of the membrane was investigated by cyclic voltammetry (CV) at self-assembled, stainless steel supported lipid bilayer membranes (s-BLMs) using a three-electrode system. The results showed that the membrane capacitance (C_m) of s-BLMs was dramatically enhanced by the presence of increasing NO concentration from 0 to 70 μM. For comparison, fullerene C₆₀ doped s-BLMs (C₆₀@s-BLMs) was also studied. The C_m of C₆₀@s-BLMs increased with NO concentration from 0 to 16 μM and gradually reached a plateau value when NO concentration was over 16 μM. We concluded that (i) NO accumulated inside lipid bilayer increases the C_m of s-BLMs, and (ii) C₆₀ inside s-BLMs changes the dielectric constant of lipid bilayer, thus reducing the effect of NO on the C_m of C₆₀@s-BLMs. This novel self-assembled lipid modified probe provides a simple yet interesting model to study the effect of NO on the electrical conductance of the membrane.     

Nonequilibrium patterns of cholesterol-rich chemical heterogenieties within single fluid supported phospholipid bilayer membranes

We have developed a simple method to introduce cholesterol- and sphingomyelin-rich chemical heterogeneities into controlled densities and concentrations within predetermined regions of another distinct fluid phospholipid bilayer supported on a solid substrate. A contiguous primary phase--a fluid POPC bilayer displaying a well-defined array of lipid-free voids (e.g., 20-100 microm squares)--was first prepared on a clean glass surface by microcontact printing under water using a poly(dimethylsiloxane) stamp. The aqueous-phase primary bilayer pattern was subsequently incubated with secondary-phase small unilamellar vesicles composed of independent chemical compositions. Backfilling by comparable vesicles resulted in gradual mixing between the primary- and secondary-phase lipids, effacing the pattern. When the secondary vesicles consisted of phase-separating mixtures of cholesterol, sphingomyelin, and a phospholipid (2:1:1 POPC/sphingomyelin/cholesterol or 1:1:1 DOPC/sphingomyelin/cholesterol), well-defined spatial patterns of fluorescence, chemical compositions, and fluidities emerged. We conjecture that these patterns form because of the differences in the equilibration rates of the secondary liquid-ordered and liquid-disordered phases with the primary fluid POPC phase. The pattern stability depended strongly on the ambient-phase temperature, cholesterol concentration, and miscibility contrast between the two phases. When cholesterol concentration in the secondary vesicles was below 20 mol %, secondary intercalants gradually diffused within the primary POPC bilayer phase, ultimately dissolving the pattern in several minutes and presumably forming a new quasi-equilibrated lipid mixture. These phase domain micropatterns retain some properties of biological rafts including detergent resistance and phase mixing induced by selective cholesterol extraction. These patterns enable direct comparisons of cholesterol- and sphingomyelin-rich phase domains and fluid phospholipid phases for their functional preferences and may be useful for developing simple, parallelized assays for phase and chemical composition-dependent membrane functionalities.     

A novel one-step method to incorporate ss DNA into bilayer lipid membranes supported on an agar electrode

A one-step method has been developed for incorporating ss DNA into a bilayer lipid membrane (BLM) on agar electrode, which displayed good responses to complementary ss DNA. The 5′-terminal phosphated end of ss DNA formed a phosphormidate bond with the amino group of N,N-diethylamidobenzene (NDAB) in the BLM forming solution. The method is easy to perform and has good reproducibility. Fourier transform infrared spectroscopy (FTIR) showed that the ss DNA was incorporated into the BLM.     

Effects of direct current bias voltages on supported bilayer lipid membranes on a glassy carbon electrode

The effects of dc bias voltages on supported bilayer lipid membranes (s-BLMs) on a glassy carbon (GC) electrode have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The experiments suggested that an appropriate positive bias voltage facilitated the formation of electrically dense membranes, but if the voltage is enough high, the lacunas on s-BLMs increased, and eventually caused the complete oxidation of the lipid membrane. While negative bias voltages could induce the damage of supported membranes to different extent. The changes of the form and quantity of graphite oxide on the surface of the electrode caused by dc bias voltages and the electroporation and damage of the membrane at high potentials may be responsible for the effects.     

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research

A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.     

Study of frictional properties of a phospholipid bilayer in a liquid environment with lateral force microscopy as a function of NaCl concentration

Friction properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-supported planar bilayers deposited on mica were tested in a liquid environment by lateral force microscopy. The presence of these bilayers was detected by imaging and force measurements with atomic force microscopy. To test how the presence of NaCl affects the frictional properties of the phospholipid bilayers, four DMPC bilayers were prepared on mica in saline media ranging from 0 to 0.1 M NaCl. Changes in the lateral vs vertical force curves were recorded as a function of NaCl concentration and related to structural changes induced in the DMPC bilayer by electrolyte ions. Three friction regimes were observed as the vertical force exerted by the tip on the bilayer increased. To relate the friction response to the structure of the DMPC bilayer, topographic images were recorded at the same time as friction data. Ions in solution screened charges present in DMPC polar heads, leading to more compact bilayers. As a consequence, the vertical force at which the bilayer broke during friction experiments increased with NaCl concentration. In addition, the topographic images showed that low-NaCl-concentration bilayers recover more easily due to the low cohesion between phospholipid molecules.     

Cannabinoid CB1 receptor recognition of endocannabinoids via the lipid bilayer: molecular dynamics simulations of CB1 transmembrane helix 6 and anandamide in a phospholipid bilayer

The phospholipid bilayer plays a central role in the lifecycle of the endogenous cannabinoid, N-arachidonoylethanolamine (anandamide, AEA). Therefore, the orientation and location of AEA in the phospholipid bilayer with respect to key membrane associated proteins, is a central issue in understanding the mechanism of endocannabinoid signaling. In this paper, we report a test of the hypothesis that a βXXβ motif (formed by beta branching amino acids, V6.43 and I6.46) on the lipid face of the cannabinoid CB1 receptor in its inactive state may serve as an initial CB1 interaction site for AEA. Eight 6 ns NAMD2 molecular dynamics simulations of AEA were conducted in a model system composed of CB1 transmembrane helix 6 (TMH6) in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer. In addition, eight 6 ns NAMD2 molecular dynamics simulations of a low CB1 affinity (20:2, n−6) analog of AEA were conducted in the same model system. AEA was found to exhibit a higher incidence of V6.43/I6.46 groove insertion than did the (20:2, n−6) analog. In certain cases, AEA established a high energy of interaction with TMH6 by first associating with the V6.43/I6.46 groove and then molding itself to the lipid face of TMH6 to establish a hydrogen bonding interaction with the exposed backbone carbonyl of P6.50. Based upon these results, we propose that the formation of this hydrogen bonded AEA/TMH6 complex may be the initial step in CB1 recognition of AEA in the lipid bilayer.     

Electric field driven changes of a gramicidin containing lipid bilayer supported on a Au(111) surface

Langmuir-Blodgett and Langmuir-Schaeffer methods were employed to deposit a mixed bilayer consisting of 90% of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 10% of gramicidin (GD), a short 15 residue ion channel forming peptide, onto a Au(111) electrode surface. This architecture allowed us to investigate the effect of the electrostatic potential applied to the electrode on the orientation and conformation of DMPC molecules in the bilayer containing the ion channel. The charge density data were determined from chronocoulometry experiments. The electric field and the potential across the membrane were determined through the use of charge density curves. The magnitudes of potentials across the gold-supported biomimetic membrane were comparable to the transmembrane potential acting on a natural membrane. The information regarding the orientation and conformation of DMPC and GD molecules in the bilayer was obtained from photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) measurements. The results show that the bilayer is adsorbed, in direct contact with the metal surface, when the potential across the interface is more positive than -0.4 V and is lifted from the gold surface when the potential across the interface is more negative than -0.4 V. This change in the state of the bilayer has a significant impact on the orientation and conformation of the phospholipid and gramicidin molecules. The potential induced changes in the membrane containing peptide were compared to the changes in the structure of the pure DMPC bilayer determined in earlier studies.