ConspectusOur current view of cellular membranes centers on the fluid-mosaic model, which envisions the cellular membrane as a “liquidlike” bilayer of lipids, cholesterol, and proteins that freely diffuse in two dimensions. In stark contrast, the exchange of materials between the leaflets of a bilayer was presumed to be prohibited by the large enthalpic barrier associated with translocating hydrophilic materials, such as a charged lipid headgroup, through the hydrophobic membrane core. This static picture with regard to lipid translocation (or “flip-flop” as it is affectionately known) has been a long-held belief in the study of membrane dynamics. The current accepted membrane model invokes specific protein flippase (inward moving), floppase (outward moving), and scramblase (bidirectional) enzymes that assist in the movement of lipids between the leaflets of cellular membranes. The low rate of protein-free lipid flip-flop has also been a cornerstone of our understanding of the bilateral organization of cellular membrane components, specifically the asymmetric distribution of lipid species found in the luminal and extracellular leaflets of the plasma membrane of eukaryotic cells.Much of the previous work contributing to our current understanding of lipid flip-flop has utilized fluorescent- or spin-labeled lipids. However, there is growing evidence that these lipid probes do not accurately convey the dynamics and thermodynamics of native (unlabeled) lipid motion. This Account summarizes our research efforts directed toward developing a deep physical and chemical understanding of protein-free lipid flip-flop in phospholipid membrane models using sum-frequency vibrational spectroscopy (SFVS). Our use of SFVS enables the direct measurement of native lipid flip-flop in model membranes. In particular, we have explored the kinetic rates and activation thermodynamics of lipid translocation as a means of deciphering the underlying chemical and physical directors governing this process. By means of transition state theory, the contributions from enthalpy and entropy on the activation energy barrier to lipid flip-flop have been explored in detail for a variety of lipid species and membrane compositions. Specifically, the effect of lipid structure and packing and the inclusion of cholesterol and transmembrane peptides on the rates and thermodynamics of lipid translocation have been investigated in detail. It is our hope that these studies will provide a new perspective on lipid translocation in biological membranes and the role of lipid flip-flop in generating and maintaining cell membrane lipid asymmetry.

The unique structure of cholesterol and its role in modulating lipid translocation (flip-flop) were examined using sum-frequency vibrational spectroscopy (SFVS). Two structural analogues of cholesterol—cholestanol and cholestene—were examined to explore the influence of ring rigidity and amphiphilicity on controlling distearoylphosphocholine (DSPC) flip-flop. Kinetic rates for DSPC flip-flop were determined as a function of sterol concentration and temperature. All three sterols increased the rate of DSPC flip-flop in a concentration-dependent manner following the order cholestene > cholestanol > cholesterol. Rates of DSPC flip-flop were used to calculate the thermodynamic activation free energy barrier (ΔG‡) in the presence of cholesterol, cholestanol, and cholestene. The acyl chain gauche content of DSPC, mean lipid area, and membrane compressibility were correlated to observed trends in ΔG‡. ΔG‡ for DSPC flip-flop showed a strong positive correlation with the molar compression modulus (K*) of the membrane, influenced by the type and concentration of the sterol added. Interestingly, cholesterol is distinctive in maintaining invariant membrane compressibility over the range of 2–10 mol %. The results in this study demonstrate that the compression modulus of a membrane plays a significant role in moderating ΔG‡ and the kinetics of native, protein-free, lipid translocation in membranes.

The kinetics and thermodynamics of 1,2-distearoyl-sn-glycero-3-[phospho(1′-rac-glycerol)] (DSPG) flip-flop in 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) membranes were examined by sum-frequency vibrational spectroscopy (SFVS). The effect of DSPG concentration in the membrane and the influence of electrolyte concentration were examined in an attempt to decipher the role the anionic PG headgroup plays in dictating the dynamics of PG flip-flop for this biologically important lipid species. DSPG flip-flop dynamics and the activation barrier to exchange were found to be directly dependent on the amount of DSPG present in the bilayer. Analysis of the activation free energy for DSPG flip-flop in mixed DSPG + DSPC bilayers reveals that charge repulsion between neighboring PG headgroups modulates the free energy barrier and subsequently, the rate of translocation. Specifically, when DSPG comprises a small portion of the bilayer, the electrostatic potential of neighboring PG lipids are effectively shielded from each other under high ionic strength conditions and little to no charge repulsion occurs. When DSPG lipids are close enough to experience charge repulsion from neighboring PG lipids, as in bilayers containing a large fraction of DSPG, or for bilayers in low ionic strength solutions, the influence of charge repulsion on the energetics of lipid flip-flop are measurable. For biological membranes, where the concentration of PG is relatively low, the neighboring PG lipids are spaced far enough apart that their anionic charges are effectively shielded, such that under physiological conditions the charged nature of the headgroup does little to modulate its lipid flip-flop energetics and corresponding rate of translocation.

The interaction of selective estrogen receptor modulators (SERMs) with lipid membranes has been measured at clinically relevant serum concentrations using the label-free technique of second harmonic generation (SHG). The SERMs investigated in this study include raloxifene, tamoxifen, and the tamoxifen metabolites 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. Equilibrium association constants (Ka) were measured for SERMs using varying lipid compositions to examine how lipid phase, packing density, and cholesterol content impact SERM-membrane interactions. Membrane-binding properties of tamoxifen and its metabolites were compared on the basis of hydroxyl group substitution and amine ionization to elucidate how the degree of drug ionization impacts membrane partitioning. SERM-membrane interactions were probed under multiple pH conditions, and drug adsorption was observed to vary with the concentration of soluble neutral species. The agreement between Ka values derived from SHG measurements of the interactions between SERMs and artificial cell membranes and independent observations of the SERMs efficacy from clinical studies suggests that quantifying membrane adsorption properties may be important for understanding SERM action in vivo.

Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 106 M–1·s–1 at 0.43 μM PnA to (1.1 ± 0.1) × 105 M–1·s–1 at 12 μM PnA. CTB–GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 109 M–1·s–1 at 0.5 nM CTB to (3.5 ± 0.2) × 106 M–1·s–1 at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 109 M–1 and (2.8 ± 0.5) × 1013 M–1, respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB–GM1 demonstrates positive ligand–ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein–ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

RuO2 conductive thin films were synthesized using the sol–gel method and deposited onto transparent insulating substrates. The optical transmission, film thickness, surface morphology and composition, resistivity, and spectroelectrochemical performance have been characterized. The optical transmission values of these films ranged from 70 to 89% in the visible region and from 56 to 88% in the infrared region. Resistivity values of the RuO2 sol–gel films varied from 1.02 × 10–3 to 1.13 Ω cm and are highly dependent on the initial solution concentration of RuO2 in the sol–gel. The RuO2 sol–gel films were used as electrodes for the electrochemical oxidation and reduction of ferrocenemethanol. The electrochemical behavior of our novel RuO2 sol–gel films was compared to that of a standard platinum disk electrode and showed no appreciable differences in the half-wave potential (E1/2). The mechanical and chemical stability of the coatings was tested by physical abrasion and exposure to highly acidic, oxidizing Piranha solution. Repeated exposure to these extreme conditions did not result in any appreciable decline in electrochemical performance. Finally, the use of the novel RuO2 sol–gel conductive and transparent films was demonstrated in a spectroelectrochemistry experiment in which the oxidation and reduction of ferrocenemethanol was monitored via UV–vis spectroscopy as the applied potential was cycled.Keywords: electrochemistry; ruthenium dioxide; sol−gel; spectroelectrochemistry; thin film; X-ray photoelectron spectroscopy;

These studies describe the implementation of second harmonic correlation spectroscopy (SHCS) to measure the adsorption and desorption kinetics of molecular species associated with a surface. Specifically, the local fluctuations of the measured second harmonic (SH) signal were used to determine the binding kinetics and thermodynamics of (S)-(+)-1,1′-bi-2-napthol SBN intercalation into a 1,2-dioleoyl-sn-glycero-3-phosphocoline (DOPC) bilayer. In order to determine the adsorption and desorption rates, the SH signal was collected above saturation concentration at steady-state equilibrium as a function of time. The autocorrelated SH signal was then fit to a correlation model developed for molecules binding at a surface when there is no contribution from molecules in solution. The measured adsorption rate for SBN to DOPC was 2.7 ± 0.2 × 103 s–1 M–1 and the desorption rate was 9 ± 4 × 10–4 s–1. The kinetic rates as well as the calculated equilibrium binding constant, 3.0 ± 1.3 × 106 M–1 obtained from SHCS were compared with those obtained from a conventional binding isotherm and found to be statistically consistent. The primary advantage of using SHCS is both the absorption and desorption rates were determined in the same experiment using only a single bulk concentration of SBN. The results of these studies demonstrate that SHCS can be used to provide accurate kinetic and thermodynamic binding data in a label-free manner in lieu of conventional isotherm studies, especially where time and analyte are scarce.

The kinetics and thermodynamics of lipid flip-flop in bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were studied using sum-frequency vibrational spectroscopy. The kinetics of DSPC and DPPS flip-flop were examined as a function of temperature and bilayer composition. The rate of DSPC flip-flop did not exhibit any significant dependence on bilayer composition while the rate of DPPS flip-flop was inversely dependent on the mole fraction of DPPS. The transition-state thermodynamics for DSPC and DPPS lipids in these mixed bilayers were determined in order to identify the energetic impact of the phosphatidylserine headgroup on lipid flip-flop. The thermodynamics for the DSPC component remained statistically identical to bilayers composed entirely of DSPC. The activation energy for the DPPS component showed a linear correlation with the mole fraction of DPPS for all bilayer compositions. The enthalpy and entropy for DPPS flip-flop did not increase linearly with the fraction of DPPS but did directly correlate with the molecular area. The DPPS component also exhibited enthalpy–entropy compensation which suggests that lipid hydration may play a significant role in membrane dynamics.

A comparison of the binding properties of avidin, streptavidin, neutrAvidin, and antibiotin antibody to a biotinylated lipid bilayer was studied using second-harmonic generation. Protein binding assays were performed on a planar supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 4 mol % biotinylated-cap-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (biotin-cap-DOPE). The equilibrium binding affinities of these biotin–protein interactions were determined, revealing the relative energetic contributions for each protein to the biotinylated lipid ligand. The results show that the binding affinities of avidin, streptavidin, and neutrAvidin for biotin were all strengthened by protein–protein interactions but that the stronger protein–protein interactions observed for streptavidin and neutrAvidin make their binding more energetically favorable. It was also shown that neutrAvidin has the highest degree of nonspecific adsorption to a pure DOPC bilayer, compared to avidin and streptavidin. In addition, the biotin-binding affinity of the antibiotin antibody was found to be of the same order of magnitude as that of avidin, streptavidin, and neutrAvidin. These findings provide important new insights into these biotin-bound protein complexes commonly used in several bioanalytical applications.

Given the complexity of cell membranes, there is a need for an analytical technique which can explore the physical properties of lipid membranes in a high-throughput and noninvasive manner. A simplified sum-frequency vibrational imaging (SFVI) setup has been developed and characterized using asymmetrically prepared 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):1,2-distearoyl(d70)-sn-glycero-3-phosphocholine (DSPC-d70) lipid bilayer arrays. Exploiting the vibrational selectivity and inherent symmetry constraints of sum-frequency generation, SFVI was successfully used to probe the transition temperature of a patterned DSPC:DSPC-d70 lipid bilayer array. SFVI was also used to study the phase behavior in a multicomponent micropatterned lipid bilayer array (MLBA) prepared using three different binary lipid mixtures (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DSPC, DOPC:1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC:DSPC). This paper demonstrates that a simplified SFVI setup provides the necessary chemical imaging capabilities with the spatial resolution, sensitivity, and field of view required for exploring lipid membrane properties in a high-throughput array based assay.

The asymmetric arrangement of phospholipids between the two leaflets of the plasma membrane of eukaryotic cells is an integral part of cellular function. ATP-dependent translocases capable of selective lipid transport across the membrane are believed to play a role in this lipid asymmetry, but our understanding of this process is incomplete. Here we show the first direct and quantitative experiments demonstrating the induction of phosphatidylserine asymmetry in a membrane by electrostatic association of poly-l-lysine in an attempt to elucidate the complex factors which govern the establishment and maintenance of lipid compositional asymmetry in the plasma membrane on a fundamental level. The attractive electrostatic interactions between the charged surface-associated polylysine and phosphatidylserine are sufficient to both induce and maintain an asymmetric arrangement of phosphatidylserine in a planar supported membrane, as measured by sum-frequency vibrational spectroscopy. These studies provide a glimpse of the physical and chemical underpinnings of lipid asymmetry in the eukaryotic plasma membrane.

Here we report the use of counter-propagating second harmonic generation (SHG) to image the interactions between the local anesthetic tetracaine and a multicomponent planar supported lipid bilayer array in a label-free manner. The lipid bilayer arrays, prepared using a 3D continuous flow microspotter, allow the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. SHG images show that tetracaine has a higher binding affinity to liquid-crystalline phase lipids than to solid-gel phase lipids. The presence of 28 mol % cholesterol decreased the binding affinity of tetracaine to bilayers composed of the mixed chain lipid, 1-steroyl-2-oleoyl-sn-glycero-3-phophocholine (SOPC), and the saturated lipids 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-dipamitoyl-sn-glycero-3-phophocholine (DPPC) while having no effect on diunsaturated 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). The maximum surface excess of tetracaine increases with the degree of unsaturation of the phospholipids and decreases with cholesterol in the lipid bilayers. The paper demonstrates that SHG imaging is a sensitive technique that can directly image and quantitatively measure the association of a drug to a multicomponent lipid bilayer array, providing a high-throughput means to assess drug–membrane interactions.

Basic transition state theory is used to describe the activation thermodynamics for phospholipid flip-flop in planar-supported lipid bilayers (PSLBs) prepared by the Langmuir−Blodgett/Langmuir−Schaeffer method. The kinetics of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) flip-flop were determined as a function of temperature and lateral surface pressure using sum-frequency vibrational spectroscopy (SFVS). From the temperature and lateral pressure dependent DSPC flip-flop kinetics, a complete description of the activation thermodynamics for flip-flop in the gel state, including free energy of activation (ΔG‡), area of activation (Δa‡), and entropy of activation (ΔS‡), was obtained. The free energy barrier for flip-flop of DSPC was determined to be ΔG‡ = 105 ± 2 kJ/mol at 40 °C at a deposition surface pressure of 30 mN/m. The free energy barrier was found to consist of large opposing entropic and enthalpic contributions. The influence of alkyl chain length on the activation thermodynamics of flip-flop was also investigated. Decreasing the alkyl chain length led to a decrease in ΔG‡ due primarily to an increase in ΔS‡. The values obtained here are compared to previous studies investigating flip-flop by vesicle based methods.

A continuous-flow microspotter was used to generate planar arrays of stabilized bilayers composed of the polymerizable lipid bis-SorbPC and dopant lipids bearing ligands for proteins. Fluorescence microscopy was used to determine the uniformity of the bilayers and to detect protein binding. After UV-initiated polymerization, poly(lipid) bilayer microarrays were air-stable. Cholera toxin subunit b (CTb) bound to an array of poly(lipid) bilayers doped with GM1, and the extent of binding was correlated to the mole percentage of GM1 in each spot. A poly(lipid) bilayer array composed of spots doped with GM1 and spots doped with biotin-DOPE specifically bound CTb and streptavidin to the respective spots from a dissolved mixture of the two proteins. Poly(bis-SorbPC)/GM1 arrays retained specific CTb binding capacity after multiple regenerations with a protein denaturing solution and also after exposure to air. In addition, these arrays are stable in vacuum, which allows the use of MALDI-TOF mass spectrometry to detect specifically bound CTb. This work demonstrates the considerable potential of poly(lipid) bilayer arrays for high-throughput binding assays and lipidomics studies.Keywords: cholera toxin; continuous-flow microspotter; GM1; MALDI-TOF MS; microarray; poly(lipid); supported lipid bilayer

In order to better characterize the dependence of lipid flip-flop rate and thermodynamics on the nature of the lipid headgroup, we have studied the kinetics of flip-flop for single-lipid and mixed-lipid bilayers consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) as a function of both pressure and temperature. The kinetics of flipping were studied by sum-frequency vibrational spectroscopy (SFVS), which does not require exogenous chemical labeling of the lipid species of interest. Additionally, SFVS may be employed to track only a single species (DSPE or DSPC) within a binary mixture by selective deuteration of the matrix lipid to make it spectroscopically inactive. Using this approach, we have found the flip-flop of pure DSPE to be slower than the flip-flop of pure DSPC by nearly 2 orders of magnitude. The thermodynamics of the pure systems were analyzed in order to better understand the physical factors underlying their transmembrane dynamics. Headgroup hydrophobicity and associated solvent effects, as well as lipid packing constraints, appear to play a key role in determining the rate of flip-flop for these two species. For mixtures of DSPE + DSPC, both components exhibited similar rates of flip-flop at a given mole fraction of DSPE. The kinetics and thermodynamics of flip-flop in the mixtures did not vary uniformly with changing composition but were well correlated to changes in the molecular packing as a function of DSPE content in the bilayer.

The influence of cholesterol (CHO) on the phase behavior of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) planar supported lipid bilayers (PSLBs) was investigated by sum-frequency vibrational spectroscopy (SFVS). The intrinsic symmetry constraints of SFVS were exploited to measure the asymmetric distribution of phase segregated phospholipid domains in the proximal and distal layers of DSPC + CHO binary mixtures as a function of CHO content and temperature. The SFVS results suggest that cholesterol significantly affects the phase segregation and domain distribution in PSLBs of DSPC in a concentration dependent manner, similar to that found in bulk suspensions. The SFVS spectroscopic measurements of phase segregation and structure change in the binary mixture indicate that membrane asymmetry must be present in order for the changes in SFVS signal to be observed. These results therefore provide important evidence for the delocalization and segregation of different phase domain structures in PSLBs due to the interaction of cholesterol and phospholipids.

We have developed a new method for creating micropatterned lipid bilayer arrays (MLBAs) using a 3D microfluidic system. An array of fluid lipid membranes was patterned onto a glass substrate using a Continuous Flow Microspotter. Fluorescence microscopy experiments were used to verify the formation of a bilayer structure on the glass substrate. Fluorescence recovery after photobleaching experiments demonstrated the bilayersʼ fluidity was maintained while being individually corralled on the substrate. The reproducibility of bilayer formation within an array was demonstrated by the linear response of membrane fluorescence versus mol % rhodamine functionalized lipids incorporated into the vesicles prior to fusion to the surface. The highly customizable nature of the MLBAs was demonstrated utilizing three different fluorescently labeled lipids to generate a multiple component lipid array. Finally, the cholera toxin B/ganglioside GM1, antidinitrophenyl (DNP) antibody/DNP, and NeutrAvidin/biotin protein−ligand systems were used to model multiple protein−ligand binding on the MLBAs. The multicomponent patterned bilayers were functionalized with GM1, DNP, and biotin lipids, and binding curves was generated by recording surface fluorescence versus increasing concentration of membrane bound ligands.

Select transmembrane proteins found in biogenic membranes are known to facilitate rapid bidirectional flip-flop of lipids between the membrane leaflets, while others have no little or no effect. The particular characteristics which determine the extent to which a protein will facilitate flip-flop are still unknown. To determine if the relative polarity of the transmembrane protein segment influences its capacity for facilitation of flip-flop, we have studied lipid flip-flop dynamics for bilayers containing the peptides WALP23 and melittin. WALP23 is used as a model hydrophobic peptide, while melittin consists of both hydrophobic and hydrophilic residues. Sum-frequency vibrational spectroscopy (SFVS) was used to characterize the bilayers and determine the kinetics of flip-flop for the lipid component, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), within the mixed bilayers. The kinetic data were utilized to determine the activation thermodynamics for DSPC flip-flop in the presence of the peptides. Melittin was found to significantly reduce the free energy barrier to DSPC flip-flop when incorporated into the bilayer at 1 mol.%, while incorporation of WALP23 at the same concentration led to a more modest reduction of the free energy barrier. The possible mechanisms by which these peptides facilitate flip-flop are analyzed and discussed in terms of the observed activation thermodynamics.

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule–membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule–membrane interactions in a high-throughput manner.Research highlights►Lipid microarrays were successfully used to model small molecule–membrane binding. ► Merocyanine 540 binding to lipid microarrays was measured using fluorescence microscopy. ► The impact of cholesterol on dye binding was dependent on the primary lipid component. ► Negatively charged lipids electrostacially repel the monomeric form of the dye from fluid phase bilayers.

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10−4 s−1 to 1.03 × 10−4 s−1) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å2/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.

The first direct experimental evidence that gramicidin A (gA), a transmembrane peptide, facilitates the translocation of unlabeled lipids in a phospholipid bilayer was obtained with sum-frequency vibrational spectroscopy (SFVS). SFVS was used to investigate the effect of gA on lipid flip-flop in a planar 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipid bilayer. The kinetics of lipid translocation were determined by an analysis of the SFVS intensity versus time at different temperatures in the presence of 2 mol % gA. The rate constants of DSPC flip-flop increase from 2 to 10 times relative to the pure DSPC system. The results indicate that facial lipid exchange can be induced by a hydrophobic transmembrane helix. The increase in lipid flip-flop rates is correlated to an increase in the gauche content of the lipid tails. The results suggest that membrane defects induced by the presence of integral membrane proteins may play a large role in modulating the rate of lipid flip-flop.