Gel-phase Bilayers Research Articles

Investigation of Transbilayer Coupling in Gel-Fluid Asymmetric Lipid Vesicles

Biological membranes display an asymmetric lipid distribution along the bilayer normal. Membrane asymmetry is also thought to affect the bilayer and the individual bilayer leaflet thicknesses. Recently, protocols enabling the preparation and characterization of stress-free, free-floating asymmetric vesicles (aLUVs) via a cyclodextrin mediated exchange were developed [1]. Here, we report of aLUVs studies with gel and fluid phase bilayer leaflets using small- and wide-angle x-ray scattering (SWAXS), and small-angle neutron scattering (SANS). Together, these techniques allow for obtaining the structural details of the two membrane leaflets. No hydrocarbon chain correlation peak was observed in the wide-angle regime of DPPC/POPC aLUVs at room temperature, consistent with an earlier observation of the partial fluidization of gel DPPC through coupling to the inner, fluid POPC leaflet [1]. On the other hand, POPE/POPC aLUVs, below the melting temperature of pure POPE, showed a WAXS-peak typical of hydrocarbons packed in a 2D hexagonal lattice. This coupling between the two bilayer leaflets manifested itself as through a small increase in area per lipid. However, our studies also showed that the underlying energetic contributions are insufficient to break up the hydrogen bond-stabilizing network formed by PE headgroups.This work is supported by the Austrian Science Fund FWF, Project No.P27083-B20 (to G.P.).[1] Heberle, F. A., Marquardt, D., Doktorova, M., Geier, B., Standaert, R. F., Heftberger, P., Kollmitzer, B., Nickels, J. D., Dick, R. A., Feigenson, G. W. et al. (2016). Langmuir, 32(20), 5195(5200).

Binding of cationic model peptides (KX)4K to anionic lipid bilayers: Lipid headgroup size influences secondary structure of bound peptides

Differential Scanning Calorimetry (DSC) and Fourier transformed Infrared (FT-IR) spectroscopy were used to test the influence of acyl chain length, acyl chain saturation, and chemical structure of anionic phospholipids on the interaction with cationic model peptides (KX)4K, with amino acid X=A, Abu, and L. The lipids used were phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and cardiolipin (CL). DSC was used to monitor the phase transition of lipid vesicles before and after peptide binding. The electrostatic attraction is the main driving force for binding. The hydrophobicity of the amino acid X influences the binding strength as well as the secondary structure of the bound peptide. Binding of peptides leads to an upshift of the lipid phase transition. Lipids with smaller headgroups show a larger upshift of the main phase transition temperature.Data from FT-IR spectroscopy show in addition that the stability of the bound β-sheets of (KX)4K depends on the hydrophobicity of the uncharged amino acid X and on the size of the lipid headgroup. For lipids with large anionic headgroups, such as PS, the antiparallel β-sheet of (KAbu)4K bound to gel phase bilayers is converted to an unordered structure upon heating through the lipid phase transition. Reducing the size of the headgroup, as in PG, increases the stability of the bound peptide β-sheets. For the smallest headgroups, present in PA and CL, stably bound β-sheets are observed even above the lipid phase transition.

Structural Properties of Phospholipid-based Bilayers with Long-Chain Alcohol Molecules in the Gel Phase.

The structural properties of two-component gel-phase bilayers of distearylphosphatidylcholine (DSPC) and alcohol molecules with different compositions and chain lengths (12-24 carbons long) are studied via molecular dynamics simulations. Several bilayer properties, including area per lipid, tilt angle, chain interdigitation, and headgroup offset, are studied for each system and compared, revealing important structural implications depending upon headgroup size and chain length. While tail tilt is the primary mechanism for single-component bilayers to balance tail attraction and headgroup repulsion, our results demonstrate that the lipid mixtures studied adjust this balance via an offset between the depths of the different molecular species in the bilayer; this behavior is found to depend both on composition and on the length of alcohol molecules relative to the length of DSPC tails. It is shown that the structural properties of bilayers with asymmetric tail lengths depend strongly on the bilayer composition, while the composition has less influence on mixed-component bilayers with nearly symmetric tail lengths. These findings are explained on the basis of the interdigitation between bilayer leaflets and how interdigitation is related to other structural properties.

Cholesterol's location in lipid bilayers

It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group is in the vicinity of the lipid–water interface. In this article we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies.

Curvature Softening and Negative Compressibility of Gel-Phase Lipid Membranes.

We show that gel-phase lipid membranes soften upon bending, leading to curvature localization and a negative compressibility. Using simulations of two very different lipid models to quantify shape and stress-strain relation of buckled membranes, we demonstrate that gel phase bilayers do not behave like Euler elastica and hence are not well described by a quadratic Helfrich Hamiltonian, much unlike their fluid-phase counterparts. We propose a theoretical framework which accounts for the observed softening through an energy density that smoothly crosses over from a quadratic to a linear curvature dependence beyond a critical new scale [Formula: see text](-1). This model captures both the shape and the stress-strain relation for our two sets of simulations and permits the extraction of bending moduli, which are found to be about an order of magnitude larger than the corresponding fluid phase values. We also find surprisingly large crossover lengths [Formula: see text], several times bigger than the bilayer thickness, rendering the exotic elasticity of gel-phase membranes more strongly pronounced than that of homogeneous compressible sheets and artificial metamaterials. We suggest that such membranes have unexpected potential as nanoscale systems with striking materials characteristics.

Molecular Simulation of the DPPE Lipid Bilayer Gel Phase: Coupling between Molecular Packing Order and Tail Tilt Angle.

The structural properties and thermal stability of dipalmitoylphosphatidylethanolamine (DPPE) in the ordered gel phase have been studied by molecular dynamics simulation using two force fields: the Berger united-atom model and the CHARMM C36 atomistic model. As is widely known, structural features are sensitive to the initial preparation of the gel phase structure, as some degrees of freedom are slow to equilibrate on the simulation time scale of hundreds of nanoseconds. In particular, we find that the degree of alignment of the lipids' glycerol backbones, which join the two hydrocarbon tails of each molecule, strongly affects the tilt angle of the tails in the resulting structures. Disorder in the backbone correlates with lower tilt angles: bilayer configurations initiated with aligned backbones produced tilt angles near 21° and 29° for the Berger and C36 force fields, respectively, while structures initiated with randomized backbone orientations showed average tilt angles of 7° and 18°, in closer agreement with the untilted structure observed experimentally. The transition temperature for the Berger force field gel bilayer has been determined by monitoring changes in width of gel phase stripe domains as a function of temperature and is 12 ± 5 K lower than the experimental value.

Effect of intra-membrane C60 fullerenes on the modulus of elasticity and the mechanical resistance of gel and fluid lipid bilayers.

Penetration and partition of C60 to the lipid bilayer core are both relevant to C60 toxicity, and useful to realise C60 biomedical potential. A key aspect is the effect of C60 on bilayer mechanical properties. Here, we present an experimental study on the mechanical effect of the incorporation of C60 into the hydrophobic core of fluid and gel phase zwitterionic phosphatidylcholine (PC) lipid bilayers. We demonstrate its incorporation inside the hydrophobic lipid core and the effect on the packing of the lipids and the vesicle size using a combination of infrared (IR) spectroscopy, atomic force microscopy (AFM) and laser light scattering. Using AFM we measured the Young's modulus of elasticity (E) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the absence (presence) of intra-membranous C60 at 24.5 °C. E of fluid phase supported bilayers is not altered by C60, but E increases with incorporation of C60 in gel phase bilayers. The increase is higher for longer hydrocarbon chains: 1.6 times for DPPC and 2 times for DSPC. However the mechanical resistance of gel phase bilayers of curved bilayered structures decreases with the incorporation of C60. Our combined results indicate that C60 causes a decrease in gel phase lipid mobility, i.e. an increase in membrane viscosity.

Membranes can Finely Tune Peptide-Induced Lipid Extraction by Modulating their Lipid Composition

Membrane binding of lytic peptides, and the resulting peptide-induced lipid extraction (membrane solubilization) are influenced by the nature of lipids forming the membranes. However, the impact of various lipid species on these two phenomena cannot be directly inferred one from the other; distinct peptide/lipid interactions are pivotal in these distinct events.Melittin is a small cationic peptide with a secondary amphipathic helical character. It interacts spontaneously with lipid bilayers, and induces lipid extraction. We investigated how phosphatidylserine (PS), cholesterol, and phosphatidylethanolamine (PE) modulate melittin binding to phosphatidylcholine (PC) membranes as well as their impact on lipid extraction.First, PS, a negatively charged phospholipids, increases melittin affinity for membranes but inhibits melittin-induced lipid efflux. It is proposed that the attractive electrostatic interactions between melittin and PS, which are responsible for the increased affinity, anchor melittin at the bilayer interface and prevent its relocation required for lipid extraction. Second, cholesterol reduces melittin membrane affinity. In parallel, it inhibits the extent of lipid extraction induced by melittin and LC-MS analysis indicates that lipid/melittin particles resulting from the extraction are cholesterol- depleted relative to the cholesterol content of the original membranes. It is proposed that the phospholipid ordering caused by cholesterol is unfavorable to peptide binding and inhibits the overall melittin action. Third, PE decreases the affinity of melittin for PC-based bilayers, an inhibition more significant in the gel than in the fluid phase. The limited amount of melittin that can be accommodated in PE-containing gel-phase bilayers appears to make the bilayers more susceptible to lipid extraction. As a consequence, PE can act as a promotor or an inhibitor of melittin-induced lipid extraction, depending on its proportion. These findings demonstrate that membranes can tune peptide-induced lipid extraction by altering their lipid composition.

Control and role of pH in peptide–lipid interactions in oriented membrane samples

To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (31P, 2H, 19F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH7, and adopts an unknown structural state at basic pH.

Structural characterization of novel cationic diC16-amidine bilayers: Evidence for partial interdigitation

In this work, the bilayer structure of novel cationic lipid diC16-amidine was compared to the one of zwitterionic dipalmitoyl phosphatidylcholine (DPPC), which shares the same hydrophobic domain.Differential scanning calorimetry shows that DPPC and diC16-amidine bilayers have similar phase transition temperatures, but diC16-amidine membranes display a less cooperative phase transition and an absence of pretransition.Both bilayers were analyzed from surface to core, using 5-, 7-, 10-, 12-, 14-, and 16-PCSL spin labels. As expected, electron spin resonance (ESR) spectra show that the gel phase of DPPC presents a flexibility gradient toward the core. In contrast, this gradient exists in the gel phase of diC16-amidine bilayers but only down to the 12th lipid tail carbon. The 14th and 16th carbons of the cationic lipid are in a very rigid environment, similar to the one observed at the bilayer surface. These data suggest that diC16-amidine molecules are organized in a partially interdigitated gel phase. ESR spectroscopy also shows that the lamellar fluid phase of diC16-amidine is more rigid than the one of DPPC.Fluorescence resonance energy transfer assays reveal that diC16-amidine displays a more efficient fusogenic activity in the gel phase than in the fluid one, suggesting that the partial interdigitation of the gel phase is important for the fusion process to occur. Since the gel–fluid transition temperature is 42°C, diC16-amidine is fusogenic at the physiological temperature and is therefore a promising lipid for delivery applications without the need of helper lipids.

Predicting proton titration in cationic micelle and bilayer environments.

Knowledge of the protonation behavior of pH-sensitive molecules in micelles and bilayers has significant implications in consumer product development and biomedical applications. However, the calculation of pKa's in such environments proves challenging using traditional structure-based calculations. Here we apply all-atom constant pH molecular dynamics with explicit ions and titratable water to calculate the pKa of a fatty acid molecule in a micelle of dodecyl trimethylammonium chloride and liquid as well as gel-phase bilayers of diethyl ester dimethylammonium chloride. Interestingly, the pKa of the fatty acid in the gel bilayer is 5.4, 0.4 units lower than that in the analogous liquid bilayer or micelle, despite the fact that the protonated carboxylic group is significantly more desolvated in the gel bilayer. This work illustrates the capability of all-atom constant pH molecular dynamics in capturing the delicate balance in the free energies of desolvation and Coulombic interactions. It also shows the importance of the explicit treatment of ions in sampling the protonation states. The ability to model dynamics of pH-responsive substrates in a bilayer environment is useful for improving fabric care products as well as our understanding of the side effects of anti-inflammatory drugs.

Systematic variation of gel-phase texture in phospholipid membranes.

The tilted gel phase of lipid bilayers can display in-plane orientational texture due to long-range alignment of the molecular director. We explore systematic variations of texture defects in a series of binary phospholipid membranes. Using polarized two-photon fluorescence microscopy, the texture pattern of single domains is revealed. The appearance of a central vortex-type defect in each domain correlates with a particular range of hydrophobic mismatch values h > 1 nm at the domain border while domains with h < 1 nm correlate with uniformly aligned texture. The central vortex defect is characterized by a defect angle, indicating its bend or splay nature. Using image analysis, we measure the defect angle and find that it has primarily bend character for small mismatch values (h ≈ 1 nm) and primarily splay nature for larger values of h. For domains containing a vortex, the domain shape is decoupled from the texture while for uniformly textured domains there is a preferred texture orientation of ≃45° along the domain border. The results establish a foundation for understanding texture phenomena in compositionally complex membranes.

Equilibrium or Quenched: Fundamental Differences between Lipid Monolayers, Supported Bilayers, and Membranes

In this work, we establish fundamental differences between the structure and packing of lipids in monolayers, supported bilayers, and multilayer films. High resolution grazing incidence X-ray diffraction reveals that monolayer structure is largely retained upon deposition onto substrates with the area per molecule controlled by deposition pressure. Such structural changes are consistent with a quenched rather than equilibrated supported membrane structure. Supported bilayers formed by vesicle fusion exhibit structural similarity to bilayers deposited at 38 mN/m, whereas packing in lipid multilayers more closely resembled bilayers deposited below 30 mN/m. At the molecular level, coupling between opposing lipid acyl chains is observed for all deposition pressures with the outer leaflet templating on the inner leaflet. Leaflet coupling induces a small condensation in the area per lipid molecule and a surprising increase in acyl chain tilt. Moreover, supported lipid bilayers exhibit preferential acyl chain alignment: the system cannot be modeled with freely rotating acyl chains as in free-standing lipid monolayers. Such acyl chain alignment is consistent with orientational texture of lipid tilt directors at larger length scales. These findings clearly demonstrate that supported, gel-phase bilayer membrane structure can be controlled and maintained by deposition onto solid supports and that increasing surface pressure induces preferential alignment of the acyl chains both within and between membrane leaflets.

Optimizing Drug Release: Bilayer to Inverted Hexagonal Phase Transition of Cationic XTC2 and Anionic DSPS Lipid System is Influenced by pH, Temperature, and Salt Concentration

The effectiveness of a macromolecular drug delivered in lipid nanoparticles (LNP) depends upon the biophysical properties of the delivery vehicle. Recent research has shown that the design of the cationic lipid component of LNPs improves the intracellular delivery of therapeutic siRNA[1]. Even in these optimized LNPs, only 1% of the siRNA taken up by the cell via endocytosis is actually released into the cell cytosol. We proposed a mechanism of endosome disruption that relies on the formation of non-bilayer phases in the presence of anionic endosomal lipid and synthetic cationic lipids. A model system using prototypical anionic lipid 1,2-distearoyl(d70)-sn-glycero-3-[phospho-L-serine] (DSPS-d70) in 1:1 molar ratio to the cationic lipid DLin-KC2-DMA (XTC2) (pKa∼6.7) was characterized by 2H and 31P NMR spectroscopy. Through spectral analysis, we determined that at physiological pH (∼7.4) the XTC2/DSPS system exhibits a stable gel phase for temperatures below 45oC while an isotropic signal emerges at higher temperatures - no inverted hexagonal (HII) phase is observed. At low pH (∼4.75), the XTC2/DSPS system is principally in a bilayer gel phase at low temperatures with a non-bilayer HII phase predominating at higher temperatures. The transition from gel to HII phase is dependent on salt concentration and is most evident in the range of 15-25oC for 0.25M [Na+], 20-30oC for 0.5M [Na+] and 35-45oC for 1M [Na+]. Through depaking the spectra, order parameter profiles SCD have been obtained and compared for DSPS-d70 chains in bilayer and HII phases. These will be useful for computational simulation and eventually to design in vivo animal model experiments.[1] Semple, S.C., et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010. 28(2): p. 172-6.

Enhancement in Lipid Bilayer Partitioning of Lysolipids and Fatty Acids Induced by their Composition

Distribution of the solutes: lysopalmitoylphosphatidylcholine (LPPC), Palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects these solutes in water. Membrane phase as well as solute concentration itself affects partitioning. Water solute concentrations were obtained at each of several bilayer lipid concentrations between 10 and 400 μM, from slopes of the linear variation of probe fluorescence properties with total solute concentrations of up to 10 % of the solvent lipid concentration. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in this range. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the solute transfer free energy were determined from the fit. Main findings are: (1) Water to bilayer transfer free energy of solute is lower for 0 to 2 % solute mole fraction than for 2 to 10 %, signaling composition induced bilayer relaxation that increases bilayer solubility, beginning at 2 % solute mole fraction. (2) Partition coefficients are in the order LPPC>PA> LPPC+PA at 37 °C. The enhanced partition coefficient of LPPC+PA signifies synergism toward increased solubility in the bilayer-gel phase. Enhancement effects were not present, where the DPPC bilayer is in the liquid phase, The observed order in the partition coefficients, at 50 °C, was LPPC≈LPPC+PA>PA. The behavior of the partition coefficients in the gel and liquid phase is similar in character to the observed presence of synergism in the transmembrane permeabilityin the bilayer gel phase and lack of it in the liquid phase. The present results provide experimental evidence that increased presence of solutes in the membrane also enhance transmembrane permeability.

Actin Assembly at Model-Supported Lipid Bilayers

We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant—making this a practical approach for creating stable lipid-actin systems via self-assembly.

Partitioning of lysolipids, fatty acids and their mixtures in aqueous lipid bilayers: Solute concentration/composition effects

Distributions of lysopalmitoylphosphatidylcholine (LPPC), palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects only the solutes in water. Water solute concentrations were obtained at each of several lipid concentrations. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in the concentration range investigated. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the free energy of transfer, for each of these solutes were determined from the fit. Main findings are: (1) Water/bilayer partition coefficient of solute is greater for 2 to 10% solute mole fraction than for 0 to 2%, signaling solute induced bilayer perturbation that increases bilayer solubility, beginning at 2% solute mole fraction. (2) Partition coefficients are in the order LPPC<PA<LPPC+PA at 37°C and LPPC+PA≤LPPC<PA at 50°C. This signifies synergism toward increased solute solubility in the bilayer-gel phase and lack of it in the bilayer-liquid phase when LPPC and PA are present together. Implications of the solute concentration/composition and bilayer phase dependences of the partition coefficients to the reported solute induced enhancements in transmembrane permeability are discussed.

Room temperature ordering of dipalmitoyl phosphatidylserine bilayers induced by short chain alcohols

Using differential scanning calorimetry and small and wide angle X-ray diffraction, we show that, following extended incubation at room temperature, methanol, propanol, and three of the isomers of butanol can induce ordering in dipalmitoyl phosphatidylserine (DPPS) gel phase bilayers. The organization of the bilayers in the presence of ethanol, described previously, is now observed to be a general effect of short chain alcohols. Evidence is presented for tilting of the acyl chains with respect to the bilayer normal in the presence of ethanol or propanol. However, the different chain lengths of the alcohols, and isomeric form, influence the thermal stability of the ordered gel to different extents. This behavior is unlike that of the gel state phosphatidylcholine analog which, in the presence of short chain alcohols, undergoes hydrocarbon chain interdigitation. Dipalmitoyl phosphatidylcholine added to DPPS in the presence of 20vol% ethanol, acts to suppress the ordered gel phase.

Examining the phase transition behavior of amphiphilic lipids in solution using statistical temperature molecular dynamics and replica-exchange Wang-Landau methods

Two different techniques - replica-exchange Wang-Landau (REWL) and statistical temperature molecular dynamics (STMD) - were applied to systematically study the phase transition behavior of self-assembling lipids as a function of temperature using an off-lattice lipid model. Both methods allow the direct calculation of the density of states with improved efficiency compared to the original Wang-Landau method. A 3-segment model of amphiphilic lipids solvated in water has been studied with varied particle interaction energies (ε) and lipid concentrations. The phase behavior of the lipid molecules with respect to bilayer formation has been characterized through the calculation of the heat capacity as a function of temperature, in addition to various order parameters and general visual inspection. The simulations conducted by both methods can go to very low temperatures with the whole system exhibiting well-ordered structures. With optimized parameters, several bilayer phases are observed within the temperature range studied, including gel phase bilayers with frozen water, mixed water (i.e., frozen and liquid water), and liquid water, and a more fluid bilayer with liquid water. The results obtained from both methods, STMD and REWL, are consistently in excellent agreement with each other, thereby validating both the methods and the results.

Lipid Bilayer Properties Control Membrane Partitioning, Binding, and Transport of P-Glycoprotein Substrates

The ABC protein P-glycoprotein (Pgp or ABCB1) is a multidrug efflux pump capable of transporting many structurally diverse substrates from within the lipid bilayer. Previous studies have demonstrated the importance of the membrane in modulating Pgp function, but few have quantified these effects. We employed purified Pgp reconstituted into phospholipid bilayers with defined gel to liquid-crystalline melting transitions to investigate the effect of membrane environment on the transporter and three of its substrates. Equilibrium dialysis measurements indicated that Hoechst 33342, LDS-751, and MK-571 partitioned much more readily into liquid-crystalline phase bilayers than into gel phase bilayers. However, drug binding affinities revealed that Pgp bound the three substrates more tightly when the lipid bilayer was in the gel phase. The binding affinity of the transporter for substrates within the bilayer was low, in the millimolar range, suggesting that it interacts with them weakly. Thermodynamic analysis revealed that both drug-Pgp and drug-lipid interactions contribute to binding affinity. The kinetics of LDS-751 and Hoechst 33342 transport by reconstituted Pgp was monitored using a real-time fluorescence-based assay to obtain apparent turnover frequencies. Transport rates were found to be sensitive to both drug structure and lipid environment. Arrhenius and transition state analysis of transport rates suggested that the rate of drug transport depends on both the affinity of Pgp for substrate and protein conformational changes. Transport rates did not appear to be limited exclusively by the rate of ATP hydrolysis and may be partially controlled by the rate of drug dissociation.