An Unexpected Driving Force for Lipid Order Appears in Asymmetric Lipid Bilayers.

An ordered phase in one leaflet of an asymmetric bilayer can induce a precisely superimposed induced order domain in the apposed leaflet. Order is induced in such simple lipid compositions as dioleoylphosphatidylcholine/cholesterol (DOPC)/chol) which is expected to be a uniform and disordered lipid mixture. Dye partitioning can be used to label and identify coexisting liquid-disordered (Ld), liquid-ordered (Lo), or gel-ordered (Lß) molecules in a phase-separated leaflet. In the other leaflet of an asymmetric bilayer, dye partitioning also labels and identifies any induced order domains created by an Lo or gel phase domain in the apposed leaflet as well as the state of disorder of the lipid surrounding the induced ordered region. We explore a molecular level mechanism by which a disorder-prone uniform mixture of DOPC/chol = 0.8/0.2 would spontaneously separate into ordered regions coexisting with disordered regions. A redistribution of cholesterol seems to take place in the regions apposed to the ordered phase. The precision of the superposition of Lo or gel domains with their induced order domains implies a strong energy penalty that would be incurred if order/disorder interfaces were to form at the bilayer midplane. We conclude that the energy penalty for Lo/Ld or gel/Ld contact in the bilayer midplane is sufficient to drive disorderly DOPC/chol into an ordered state that reduces unfavorable order-disorder contacts at the bilayer midplane interface.

Nano-scale domains in the plasma membrane are like macroscopic domains in asymmetric bilayers.

Unfavorable lipid-lipid pairwise interactions between HiTm and LowTm lipids drive liquid-disordered (Ld) + liquid-ordered (Lo) phase separation. Large size of phase domains is opposed by lipid dipole repulsions, which are more significant compared with the pairwise interactions for naturally abundant LowTm lipids such as palmitoyl oleoyl phosphatidylcholine. During the nano-to-macro domain size transition, no lipid phase transition occurs, and measured properties of Ld + Lo nanodomains are found to be essentially the same as those of macrodomains. Use of macrodomains in mixtures to model cell plasma membranes (PM) is helpful, enabling study by optical microscopy. Use of asymmetric giant unilamellar vesicles to model a PM reveals that ordered phase domains in one leaflet induce ordered domains in an otherwise uniform phase in the apposing leaflet that models a cytoplasmic leaflet. Because macro and nano phase properties are so similar, we conclude that a cell PM that has nano-scale Ld + Lo phase domains in the exoplasmic leaflet is likely to induce nano-scale ordered domains in the cytoplasmic leaflet.

Improving our picture of the plasma membrane: Rafts induce ordered domains in a simplified model cytoplasmic leaflet.

By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion. aGUVs were detached from the SLB for study. In general these aGUVs are formed with one leaflet that phase-separates into Ld (liquid disordered) + Lo (liquid ordered) phases, and another leaflet with lipid composition that would form a single fluid phase in a symmetric bilayer. We observed that ordered phases of either Lo or Lß (gel phase) induce an ordered domain in the apposed fluid leaflet that lacks high melting lipids. Results suggest both an inter-leaflet and an intra-leaflet redistribution of cholesterol. We used C-Laurdan spectral images to investigate the lipid packing/order of aGUVs, finding that cholesterol partitions into the induced ordered domains. We suggest this behavior to be commonplace, that when Ld + Lo phase separation occurs in a cell PM exoplasmic leaflet, an induced order domain forms in the cytoplasmic leaflet.

On the small size of liquid-disordered + liquid-ordered nanodomains.

Four-component phase diagrams reveal that Liquid-disordered + liquid-ordered (Ld + Lo) nanodomains are exclusively found adjacent to a three-phase region, and so cannot be a one-phase microemulsion. Of importance for understanding biological membranes, a small change in lipid bilayer composition can change the size of these coexisting phase domains hundreds of fold, between tens of nanometers and microns. Nanodomain diameter, measured from small angle neutron scattering, is in the range 15-35 nm, consistent with stabilization by repulsive dipole fields. Ld/Lo line tension controls the Ld + Lo domain size transition. Other than size, chemical and physical properties of the phase domains do not seem to change during the transition. Unfavorable lipid-lipid pairwise interactions, rather than phase thickness mismatch, seem to be the main reason for Ld + Lo immiscibility. Pairwise interactions of cholesterol-phospholipid seem to be favorable, whereas pairwise interactions of high-melting phospholipid with low-melting phospholipid are unfavorable. Measured Ld/Lo line tension, like the phase separation, is created mainly by unfavorable lipid-lipid pairwise interactions. Lipid dipole-dipole repulsion opposes these unfavorable lipid-lipid pairwise interactions and thus, in a sense, is the reason that nanodomains form. Bilayer physical and chemical properties measured from macroscopic domains of coexisting Ld + Lo phases should be good approximations for the properties of coexisting nanoscopic domains.

Dataset of asymmetric giant unilamellar vesicles prepared via hemifusion: Observation of anti-alignment of domains and modulated phases in asymmetric bilayers.

The data provided with this paper are confocal fluorescence images of symmetric giant unilamellar vesicles (GUVs) and asymmetric giant unilamellar vesicles (aGUVs). In this work, aGUVs were prepared using the hemifusion method and are labelled with two different fluorescent dyes, named TFPC and DiD. Both dyes show strong preference for the liquid-disordered (Ld) phase instead of the liquid-ordered (Lo) phase. The partition of these dyes favoring the Ld phase leads to bright Ld phase and dark Lo phase domains in symmetric GUVs observed by fluorescence microscopy. In symmetric vesicles, the bright and the dark domains of the inner and the outer leaflets are aligned. In aGUVs, the fluorescent probe TFPC exclusively labels the aGUV outer leaflet. Here, we show a dataset of fluorescence micrographs obtained using scanning fluorescence confocal microscopy. For the system chosen, the fluorescence signal of TFPC and DiD show anti-alignment of the brighter domains on aGUVs. Important for this dataset, TFPC and DiD have fluorescence emission centered in the green and far-red region of the visible spectra, respectively, and the dyes' fluorescence emission bands do not overlap. This dataset were collected in the same conditions of the dataset reported in the co-submitted work (Enoki, et al. 2021) where most of aGUVs show domains alignment. In addition, we show micrographs of GUVs displaying modulated phases and macrodomains. We also compare the modulated phases observed in GUVs and aGUVs. For these datasets, we collected a sequence of micrographs using confocal microscopy varying the z-position, termed a z-stack. Images were collected in a scanning microscope Nikon Eclipse C2+ (Nikon Instruments, Melville, NY). Additional samples used to measure the lipid concentrations and to prepare GUVs with accurate lipid fractions are also provided with this paper.

Investigation of the domain line tension in asymmetric vesicles prepared via hemifusion.

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase. To better understand the interleaflet interactions that influence domains, we compared asymmetric model membranes that capture salient properties of the PM with simpler symmetric membranes. Using asymmetric giant unilamellar vesicles (aGUVs) prepared by hemifusion with a supported lipid bilayer, we investigate the domain line tension that characterizes the behavior of coexisting ordered + disordered domains. The line tension can be related to the contact perimeter of the different phases. Compared to macroscopic phase separation, the appearance of modulated phases was found to be a robust indicator of a decrease in domain line tension. Symmetric GUVs of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/cholesterol (chol) were formed into aGUVs by replacing the GUV outer leaflet with DOPC/chol = 0.8/0.2 in order to create a cytoplasmic leaflet model. These aGUVs revealed lower line tension for the ordered + disordered domains of the exoplasmic model leaflet.

PI(4,5)P2 Clustering and Its Impact on Biological Functions.

Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P2] composes only 1-2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P2 recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P2 in membranes. Physiological multivalent cations such as Ca2+ and Mg2+ can bridge between PI(4,5)P2 headgroups, forming nanoscopic PI(4,5)P2-cation clusters. The distinct lipid environment surrounding PI(4,5)P2 affects the degree of PI(4,5)P2 clustering. In addition, diverse cellular proteins interacting with PI(4,5)P2 can further regulate PI(4,5)P2 lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P2 behavior in both cells and model membranes, with emphasis on both multivalent cation- and protein-induced PI(4,5)P2 clustering. Understanding the nature of spatially separated pools of PI(4,5)P2 is fundamental to cell biology.

Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition.

The eukaryotic plasma membrane (PM) exhibits lipid mixing heterogeneities known as lipid rafts. These lipid rafts, the result of liquid-liquid phase separation, can be modeled by coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. Four-lipid component systems with a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol) can give rise to domains of different sizes. These four-component systems have been characterized in experiments, yet there are few studies that model the asymmetric distribution of lipids actually found in the PM. We used molecular dynamics (MD) simulations to analyze the transition from nanoscopic to macroscopic domains in symmetric and in asymmetric model membranes. Using coarse-grained MD simulations, we found that asymmetry promotes macroscopic domain growth in a case where symmetric systems exhibit nanoscopic domains. Also, macroscopic domain formation in symmetric systems is highly dependent on registration of like phases in the cytoplasmic and exoplasmic leaflets. Using united-atom MD simulations, we found that symmetric Lo domains are only slightly more ordered than asymmetric Lo domains. We also found that large Lo domains in our asymmetric systems induce a slight chain ordering in the apposed cytoplasmic regions. The chol fractions of phase-separated Lo and Ld domains of the exoplasmic leaflet were unchanged whether the system was symmetric or asymmetric.

Mechanisms of PI(4,5)P2 Enrichment in HIV-1 Viral Membranes.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for HIV-1 virus assembly. The viral membrane is enriched in PIP2, suggesting that the virus assembles at PIP2-rich microdomains. We showed previously that in model membranes PIP2 can form nanoscopic clusters bridged by multivalent cations. Here, using purified proteins we quantitated the binding of HIV-1 Gag-related proteins to giant unilamellar vesicles containing either clustered or free PIP2. Myristoylated MA strongly preferred binding to clustered PIP2. By contrast, unmyristoylated HIV-1 MA, RSV MA, and a PH domain all preferred to interact with free PIP2. We also found that HIV-1 Gag multimerization promotes PIP2 clustering. Truncated Gag proteins comprising the MA, CA, and SP domains (MACASP) or the MA and CA domains (MACA) induced self-quenching of acyl chain-labeled fluorescent PIP2 in liposomes, implying clustering. However, HIV-1 MA itself did not induce PIP2 clustering. A CA inter-hexamer dimer interface mutation led to a loss of induced PIP2 clustering in MACA, indicating the importance of protein multimerization. Cryo-electron tomography of liposomes with bound MACA showed an amorphous protein layer on the membrane surface. Thus, it appears that while protein-protein interactions are required for PIP2 clustering, formation of a regular lattice is not. Protein-induced PIP2 clustering and multivalent cation-induced PIP2 clustering are additive. Taken together, these results provide the first evidence that HIV-1 Gag can selectively target pre-existing PIP2-enriched domains of the plasma membrane for viral assembly, and that Gag multimerization can further enrich PIP2 at assembly sites. These effects could explain the observed PIP2 enrichment in HIV-1.

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors.

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Molecular Dynamics Simulations Reveal Leaflet Coupling in Compositionally Asymmetric Phase-Separated Lipid Membranes.

The eukaryotic plasma membrane has an asymmetric distribution of its component lipids. Rafts that result from liquid-liquid phase separation are a feature of its exoplasmic leaflet, but how these exoplasmic leaflet domains are coupled to the cytoplasmic leaflet is not understood. These rafts can be studied in model membranes of three-component mixtures that produce coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. We conducted all-atom molecular dynamics simulations of compositionally asymmetric lipid bilayers that reflect a more realistic model of the plasma membrane. One leaflet contained phase-separated domains with phosphatidylcholine and cholesterol, representing the exoplasmic leaflet, whereas the other contained phosphatidylethanolamine, phosphatidylserine, and cholesterol, which are the predominant components of the cytoplasmic leaflet. Inspired by findings of domain alignment across the two leaflets in compositionally symmetric model membranes, we examined the coupling between the two leaflets to see how the single-phase cytoplasmic leaflet would respond to phase separation in the other leaflet and if information could be communicated across the membrane. We found the region of the single-phase leaflet apposing the Lo domain to be slightly more ordered and thicker than the region apposing the Ld domain. The region across from the Lo domain is somewhat enriched in cholesterol and significantly depleted of polyunsaturated lipids.

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles.

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes.

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld) + liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld + Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.

Presence and Role of Midplane Cholesterol in Lipid Bilayers Containing Registered or Antiregistered Phase Domains.

Three-component lipid mixtures can produce coexisting liquid ordered and liquid disordered phases, a model for eukaryotic plasma membrane rafts. In compositionally symmetric bilayers with two phase-separated leaflets, phase domains of the two leaflets may align through registration, where domains are found across from domains of the same phase, or else antiregistration, where domains are found across from domains of the opposite phase. This alignment could serve as a method of information communication across the plasma membrane. We used coarse-grained molecular dynamics simulations to study ternary mixtures of a high-melting-temperature phospholipid, a low-melting-temperature phospholipid, and cholesterol. We found a significant presence of cholesterol molecules at the bilayer midplane rather than in a leaflet in some systems, corresponding to a lack of registration. Increasing the length of the acyl chains from 16 to 24 carbons in high-melting-temperature phospholipids or increasing the concentration of cholesterol from 20 to 35 mol % in the bilayer produced a transition from registration to antiregistration and gave rise to significant populations of midplane cholesterol.

Multivalent Cation-Bridged PI(4,5)P2 Clusters Form at Very Low Concentrations.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca2+, Mg2+, Zn2+, or trivalent ions Fe3+ and Al3+. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases.

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (∼200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5-10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ∼5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.