Wetting and capillary condensation as means of protein organization in membranes.

Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers.

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.     

Topology and lipid selectivity of pulmonary surfactant protein SP-B in membranes: Answers from fluorescence

Contradictory results have been reported with respect to the depth of penetration and the orientation of pulmonary surfactant protein SP-B in phospholipid membranes and its relative selectivity to interact with anionic over zwitterionic phospholipid species. In the present study we have re-evaluated lipid-protein interactions of SP-B by analysing F?rster resonance energy transfer (FRET) efficiencies, obtained from time-resolved measurements, from the single tryptophan in SP-B to different fluorescently labelled phospholipids in matrix bilayers made of either pure phosphatidylcholine (POPC) or the full lipid extract obtained from purified surfactant. In the background of POPC membranes SP-B exhibits a certain level of selectivity for anionic fluorescent phospholipids over the corresponding zwitterionic analogues, but apparently no preference for phosphatidylglycerol over other anionic species such as phosphatidylserine. No selectivity was detected in membranes made of full surfactant lipids, indicating that specific lipid-protein binding sites could already be occupied by endogenous anionic phospholipids. Furthermore, we have analysed the fit of two different models of how SP-B could be orientated with respect to phospholipid membrane surfaces to the FRET data. The FRET results are consistent with topology models in which the protein has a superficial orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction. This discards a deep penetration of the protein into the core of bilayers and suggests that most hydrophobic segments of SP-B could participate in protein-protein instead of lipid-protein interactions.     

Model answers to lipid membrane questions

Ever since it was discovered that biological membranes have a core of a bimolecular sheet of lipid molecules, lipid bilayers have been a model laboratory for investigating physicochemical and functional properties of biological membranes. Experimental and theoretical models help the experimental scientist to plan experiments and interpret data. Theoretical models are the theoretical scientist's preferred toys to make contact between membrane theory and experiments. Most importantly, models serve to shape our intuition about which membrane questions are the more fundamental and relevant ones to pursue. Here we review some membrane models for lipid self-assembly, monolayers, bilayers, liposomes, and lipid-protein interactions and illustrate how such models can help answering questions in modern lipid cell biology.     

Lipid enrichment and selectivity of integral membrane proteins in two-component lipid bilayers

A model recently used to study lipid-protein interactions in one-component lipid bilayers (Sperotto and Mouritsen, 1991 a, b) has been extended in order to include two different lipid species characterized by different acyl-chain lengths. The model, which is a statistical mechanical lattice model, assumes that hydrophobic matching between lipid-bilayer hydrophobic thickness and hydrophobic length of the integral protein is an important aspect of the interactions. By means of Monte Carlo simulation techniques, the lateral distribution of the two lipid species near the hydrophobic protein-lipid interface in the fluid phase of the bilayer has been derived. The results indicate that there is a very structured and heterogeneous distribution of the two lipid species near the protein and that the protein-lipid interface is enriched in one of the lipid species. Out of equilibrium, the concentration profiles of the two lipid species away from the protein interface are found to develop a long-range oscillatory behavior. Such dynamic membrane heterogeneity may be of relevance for determining the physical factors involved in lipid specificity of protein function.     

Formation and regulation of lipid microdomains in cell membranes: Theory, modeling, and speculation

Compositional lipid microdomains (“lipid rafts”) in plasma membranes are believed to be important components of many cellular processes. The biophysical mechanisms by which cells regulate the size, lifetime, and spatial localization of these domains are rather poorly understood at the moment. Over the years, experimental studies of raft formation have inspired several phenomenological theories and speculations incorporating a wide variety of thermodynamic assumptions regarding lipid-lipid and lipid-protein interactions, and the potential role of active cellular processes on membrane structure. Here we critically review and discuss these theories, models, and speculations, and present our view on future directions.     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

Simulation studies of protein-induced bilayer deformations, and lipid-induced protein tilting, on a mesoscopic model for lipid bilayers with embedded proteins

Biological membranes are complex and highly cooperative structures. To relate biomembrane structure to their biological function it is often necessary to consider simpler systems. Lipid bilayers composed of one or two lipid species, and with embedded proteins, provide a model system for biological membranes. Here we present a mesoscopic model for lipid bilayers with embedded proteins, which we have studied with the help of the dissipative particle dynamics simulation technique. Because hydrophobic matching is believed to be one of the main physical mechanisms regulating lipid-protein interactions in membranes, we considered proteins of different hydrophobic length (as well as different sizes). We studied the cooperative behavior of the lipid-protein system at mesoscopic time- and lengthscales. In particular, we correlated in a systematic way the protein-induced bilayer perturbation, and the lipid-induced protein tilt, with the hydrophobic mismatch (positive and negative) between the protein hydrophobic length and the pure lipid bilayer hydrophobic thickness. The protein-induced bilayer perturbation was quantified in terms of a coherence length, xi(P), of the lipid bilayer hydrophobic thickness profile around the protein. The dependence on temperature of xi(P), and the protein tilt-angle, were studied above the main-transition temperature of the pure system, i.e., in the fluid phase. We found that xi(P) depends on mismatch, i.e., the higher the mismatch is, the longer xi(P) becomes, at least for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting (or overshooting) region where the bilayer hydrophobic thickness is locally lower (or higher) than in the unperturbed bilayer, depending on whether the protein hydrophobic length is longer (or shorter) than the pure lipid bilayer hydrophobic thickness. Proteins may tilt when embedded in a too-thin bilayer. Our simulation data suggest that, when the embedded protein has a small size, the main mechanism to compensate for a large hydrophobic mismatch is the tilt, whereas large proteins react to negative mismatch by causing an increase of the hydrophobic thickness of the nearby bilayer. Furthermore, for the case of small, peptidelike proteins, we found the same type of functional dependence of the protein tilt-angle on mismatch, as was recently detected by fluorescence spectroscopy measurements.     

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.     

Lipids do influence protein function-the hydrophobic matching hypothesis revisited

A topical review of the current state of lipid-protein interactions is given with focus on the physical interactions between lipids and integral proteins in lipid-bilayer membranes. The concepts of hydrophobic matching and curvature stress are revisited in light of recent data obtained from experimental and theoretical studies which demonstrate that not only do integral proteins perturb the lipids, but the physical state of the lipids does also actively influence protein function. The case of the trans-membrane water-channel protein aquaporin GlpF from E. coli imbedded in lipid-bilayer membranes is discussed in some detail. Numerical data obtained from Molecular Dynamics simulations show on the one side that the lipid bilayer adapts to the channel by a hydrophobic matching condition which reflects the propensity of the lipid molecules for forming curved structures. On the other side, it is demonstrated that the transport function of the channel is modulated by the matching condition and/or the curvature stress in a lipid-specific manner.     

Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Real-time imaging of lipid domains and distinct coexisting membrane protein clusters

A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid-protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14-47μm), protein clusters exhibited a narrow size distribution (2.6-4.8μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid-protein interactions on lateral membrane architecture.     

Theoretical analysis of hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated protein-protein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 A without gramicidin to 32.7 A. In contrast, the same molar ratio of gramicidin in DLPC increases the PtP from 30.8 A to 32.1 A. Concurrently, x-ray in-plane scattering showed that the most probable nearest-neighbor separation between gramicidin channels was 26.8 A in DLPC, but reduced to 23.3 A in DMPC. In this paper we review the idea of hydrophobic matching in which the lipid bilayer deforms to match the hydrophobic surface of the embedded proteins. We use a simple elasticity theory, including thickness compression, tension, and splay terms to describe the membrane deformation. The energy of membrane deformation is compared with the energy cost of hydrophobic mismatch. We discuss the boundary conditions between a gramicidin channel and the lipid bilayer. We used a numerical method to solve the problem of membrane deformation profile in the presence of a high density of gramicidin channels and ran computer simulations of 81 gramicidin channels to find the equilibrium distributions of the channels in the plane of the bilayer. The simulations contain four parameters: bilayer thickness compressibility 1/B, bilayer bending rigidity Kc, the channel-bilayer mismatch Do, and the slope of the interface at the lipid-protein boundary s. B, Kc, and Do were experimentally measured; the only free parameter is s. The value of s is determined by the requirement that the theory produces the experimental values of bilayer thinning by gramicidin and the shift in the peak position of the in-plane scattering due to membrane-mediated channel-channel interactions. We show that both hydrophobic matching and membrane-mediated interactions can be understood by the simple elasticity theory.     

Binding of prion proteins to lipid membranes

A key molecular event in prion diseases is the conversion of the normal cellular form of the prion protein (PrPC) to an aberrant form known as the scrapie isoform, PrPSc. Under normal physiological conditions PrPC is attached to the outer leaflet of the plasma membrane via a GPI-anchor. It has been proposed that a direct interaction between PrP and lipid membranes could be involved in the conversion of PrPC to its disease-associated corrupted conformation, PrPSc. Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of recombinant PrP isoforms to model lipid membranes using surface plasmon resonance spectroscopy. The binding of alpha- and beta-PrP to negatively charged lipid membranes of POPG, zwitterionic membranes of DPPC, and model raft membranes composed of DPPC, cholesterol, and sphingomyelin is compared at pH 7 and 5, to simulate the environment at the plasma membrane and within endosomes, respectively. It is found that PrP binds strongly to lipid membranes. The strength of the association of PrP with lipid membranes depends on the protein conformation and pH, and involves both hydrophobic and electrostatic lipid-protein interactions. Competition binding measurements established that the binding of alpha-PrP to lipid membranes follows a decreasing order of affinity to POPG>DPPC>rafts.     

Lipid-protein interactions and the function of the Ca2+-ATPase of sarcoplasmic reticulum

Regardless of the nature of the protein constituents of membranes, the molecular arrangement of lipids interacting with them must satisfy hydrophobic, ionic, and steric requirements. Biological membranes have a great diversity of lipid constituents, and this diversity might have functional roles. It has been proposed, for example, that the hydrophobic regions of membrane proteins are stabilized in the membrane through interactions with lipids able to adopt configurations other than the bilayer structure. Progress in understanding at the molecular level how lipid-protein interactions control the properties of membrane proteins has been hindered by the lack of information concerning the structure of the hydrophobic regions of membrane proteins. Nevertheless, there are many examples in the literature describing how changes in the lipid environment affect physical and biochemical properties of membrane proteins. From these studies, discussed in this review, an overall picture of how lipids and proteins interact in membranes is beginning to emerge.