Lipid polymorphism and biomembrane function

In recent years, several major developments have taken place in the biology, physical chemistry and technology of polymorphism of membrane lipids. These include the identification of polymorphic regulation of membrane lipid composition in Escherichia coli, the importance of nonbilayer lipids for protein functioning, the special packing properties of bilayers containing these lipids, and the crystalization of a membrane protein out of three dimensional bilayer networks (lipid cubic phases). These exciting developments bring us closer to understanding the paradox of the lipid bilayer structure of biomembranes and the molecular basis of membrane protein structure and function.     

An N-terminal Amphipathic Helix Binds Phosphoinositides and Enhances Kalirin Sec14 Domain-mediated Membrane Interactions

Previous studies revealed an essential role for the lipid-binding Sec14 domain of kalirin (KalSec14), but its mechanism of action is not well understood. Because alternative promoter usage appends unique N-terminal peptides to the KalSec14 domain, we used biophysical, biochemical, and cell biological approaches to examine the two major products, bKalSec14 and cKalSec14. Promoter B encodes a charged, unstructured peptide, whereas promoter C encodes an amphipathic helix (Kal-C-helix). Both bKalSec14 and cKalSec14 interacted with lipids in PIP strip and liposome flotation assays, with significantly greater binding by cKalSec14 in both assays. Disruption of the hydrophobic face of the Kal-C-helix in cKalSec14_KKED eliminated its increased liposome binding. Although cKalSec14 showed significantly reduced binding to liposomes lacking phosphatidylinositol phosphates or cholesterol, liposome binding by bKalSec14 and cKalSec14_KKED was not affected. When expressed in AtT-20 cells, bKalSec14-GFP was diffusely localized, whereas cKalSec14-GFP localized to the trans-Golgi network and secretory granules. The amphipathic C-helix was sufficient for this localization. When AtT-20 cells were treated with a cell-permeant derivative of the Kal-C-helix (Kal-C-helix-Arg₉), we observed increased secretion of a product stored in mature secretory granules, with no effect on basal secretion; a cell-permeant control peptide (Kal-C-helix_KKED-Arg₉) did not have this effect. Through its ability to control expression of a novel, phosphoinositide-binding amphipathic helix, Kalrn promoter usage is expected to affect function.     

Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes

Membranes lipids are one of the most adaptable molecules in response to perturbations. Even subtle changes of the composition of acyl chains or head groups can alter the packing arrangements of lipids within the bilayer. This changes the balance between bilayer and nonbilayer lipids, serving to affect bilayer stability and fluidity, as well as altering lipid-protein interactions. External factors can also change membrane fluidity and lipid composition; including temperature, chemicals, ions, pressure, nutrients and the growth phase of the microbial culture. Various biophysical techniques have been used to monitor fluidity changes within the bacterial membrane. In this review, bacterial cytoplasmic membrane changes and related functional effects will be examined as well as the use of fluorescence polarization methods and examples of data obtained from research with bacteria.     

Effect of nonbilayer lipids on membrane binding and insertion of the catalytic domain of leader peptidase

Biological membranes contain a substantial amount of "nonbilayer lipids", which have a tendency to form nonlamellar phases. In this study the hypothesis was tested that the presence of nonbilayer lipids in a membrane, due to their overall small headgroup, results in a lower packing density in the headgroup region, which might facilitate the interfacial insertion of proteins. Using the catalytic domain of leader peptidase (delta2-75) from Escherichia coli as a model protein, we studied the lipid class dependence of its insertion and binding. In both lipid monolayers and vesicles, the membrane binding of (catalytically active) delta2-75 was much higher for the nonbilayer lipid DOPE compared to the bilayer lipid DOPC. For the nonbilayer lipids DOG and MGDG a similar effect was observed as for DOPE, strongly suggesting that no specific interactions are involved but that the small headgroups create hydrophobic interfacial insertion sites. On the basis of the results of the monolayer experiments, calculations were performed to estimate the space between the lipid headgroups accessible to the protein. We estimate a maximal size of the insertion sites of 15 +/- 7 A2/lipid molecule for DOPE, relative to DOPC. The size of the insertion sites decreases with an increase in headgroup size. These results show that nonbilayer lipids stimulate the membrane insertion of delta2-75 and support the idea that such lipids create insertion sites by reducing the packing density at the membrane-water interface. It is suggested that PE in the bacterial membrane facilitates membrane insertion of the catalytic domain of leader peptidase, allowing the protein to reach the cleavage site in preproteins.     

Clustering of α-Synuclein on Supported Lipid Bilayers: Role of Anionic Lipid, Protein, and Divalent Ion Concentration

α-Synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that α-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of α-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques. The membranes contained phosphatidylcholine and phosphatidylglycerol. In the absence of protein, these lipids mix uniformly. Our results show that the propensity of α-synuclein to cluster on the membrane increases as the concentration of anionic lipid and/or protein increases. Regions on the lipid bilayer where α-synuclein is clustered are enriched in phosphatidylglycerol. We also observe divalent metal ions stimulate protein cluster formation, primarily by promoting lipid demixing. The importance of protein structure, lipid demixing, and divalent ions, as well as the physiological implications, will be discussed. Because membrane-bound α-synuclein assemblies may play a role in neurotoxicity, it is of interest to determine how membranes can be used to tune the propensity of α-synuclein to aggregate.     

In vitro Unfolding and Refolding of the Small Multidrug Transporter EmrE

The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process. EmrE is part of a multidrug resistance protein family that is highly conserved amongst bacteria and is responsible for bacterial resistance to toxic substances. We find that the secondary structure of EmrE is very stable and only small amounts are denatured even in the presence of unusually high denaturant concentrations involving a combination of 10 M urea and 5% SDS. Substrate binding by EmrE is recovered after refolding this denatured protein into dodecylmaltoside detergent micelles or into lipid vesicles. The yield of refolded EmrE decreases with lipid bilayer compositional changes that increase the lateral chain pressure within the bilayer, whilst conversely, the apparent rate of folding seems to increase. These results add further weight to the hypothesis that an increased lateral chain pressure hinders protein insertion across the bilayer. Once the protein is inserted, however, the greater pressure on the transmembrane helices accelerates correct packing and final folding. This work augments the relatively small number of biophysical folding studies in vitro on helical membrane proteins.     

Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ～9–14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5–14.5 Å) for W4 insertion.     

The hydrophobic insertion mechanism of membrane curvature generation by proteins

A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices.     

Specific Membrane Binding of Neurotoxin II Can Facilitate Its Delivery to Acetylcholine Receptor

The action of three-finger snake α-neurotoxins at their targets, nicotinic acetylcholine receptors (nAChR), is widely studied because of its biological and pharmacological relevance. Most such studies deal only with ligands and receptor models; however, for many ligand/receptor systems the membrane environment may affect ligand binding. In this work we focused on binding of short-chain α-neurotoxin II (NTII) from Naja oxiana to the native-like lipid bilayer, and the possible role played by the membrane in delivering the toxin to nAChR. Experimental (NMR and mutagenesis) and molecular modeling (molecular-dynamics simulation) studies revealed a specific interaction of the toxin molecule with the phosphatidylserine headgroup of lipids, resulting in the proper topology of NTII on lipid bilayers favoring the attack of nAChR. Analysis of short-chain α-neurotoxins showed that most of them possess a high positive charge and sequence homology in the lipid-binding motif of NTII, implying that interaction with the membrane surrounding nAChR may be common for the toxin family.     

Contribution of Membrane Elastic Energy to Rhodopsin Function

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the ²H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.     

The activity of the amphipathic peptide delta-lysin correlates with phospholipid acyl chain structure and bilayer elastic properties

Release of lipid vesicle content induced by the amphipathic peptide δ-lysin was investigated as a function of lipid acyl chain length and degree of unsaturation for a series of phosphatidylcholines. Dye efflux and peptide binding were examined for three homologous lipid series: di-monounsaturated, di-polyunsaturated, and asymmetric phosphatidylcholines, with one saturated and one monounsaturated acyl chain. Except for the third series, peptide activity correlated with the first moment of the lateral pressure profile, which is a function of lipid acyl chain structure. In vesicles composed of asymmetric phosphatidylcholines, peptide binding and dye efflux are enhanced compared to symmetric, unsaturated lipids with similar pressure profiles. We attribute this to the entropically more favorable interaction of δ-lysin with partially saturated phospholipids. We find that lipid acyl chain structure has a major impact on the activity of δ-lysin and is likely to be an important factor contributing to the target specificity of amphipathic peptides.     

Binding of Aβ peptide creates lipid density depression in DMPC bilayer

Using isobaric–isothermal replica exchange molecular dynamics and all-atom explicit water model we study the impact of Aβ monomer binding on the equilibrium properties of DMPC bilayer. We found that partial insertion of Aβ peptide into the bilayer reduces the density of lipids in the binding “footprint” and indents the bilayer thus creating a lipid density depression. Our simulations also reveal thinning of the bilayer and a decrease in the area per lipid in the proximity of Aβ. Although structural analysis of lipid hydrophobic core detects disordering in the orientations of lipid tails, it also shows surprisingly minor structural perturbations in the tail conformations. Finally, partial insertion of Aβ monomer does not enhance water permeation through the DMPC bilayer and even causes considerable dehydration of the lipid–water interface. Therefore, we conclude that Aβ monomer bound to the DMPC bilayer fails to perturb the bilayer structure in both leaflets. Limited scope of structural perturbations in the DMPC bilayer caused by Aβ monomer may constitute the molecular basis of its low cytotoxicity.     

Bilayer interfacial properties modulate the binding of amphipathic peptides

The free energy of transfer (DeltaG degrees ) from water to lipid bilayers was measured for two amphipathic peptides, the presequence of the mitochondrial peptide rhodanese (MPR) and melittin. Experiments were designed to determine the effects on peptide partitioning of the addition of lipids that produce structural modifications to the bilayer/water interface. In particular, the addition of cholesterol or the cholesterol analog 6-ketocholestanol increases the bilayer area compressibility modulus, indicating that these molecules modify lipid-lipid interactions in the plane of the bilayer. The addition of 6-ketocholestanol or lipids with attached polyethylene glycol chains (PEG-lipids) modify the effective thickness of the interfacial region; 6-ketocholestanol increases the width of hydrophilic headgroup region in the direction of the acyl chains whereas the protruding PEG chains of PEG-lipids increase the structural width of the headgroup region into the surrounding aqueous phase. The incorporation of PEG-lipids with PEG molecular weights of 2000 or 5000 had no appreciable effect on peptide partitioning that could not be accounted for by the presence of surface charge. However, for both MPR and melittin DeltaG degrees decreased linearly with increasing bilayer compressibility modulus, demonstrating the importance of bilayer mechanical properties in the binding of amphipathic peptides.     

Effects of curvature and composition on α-synuclein binding to lipid vesicles

Parkinson's disease is characterized by the presence of intracellular aggregates composed primarily of the neuronal protein α-synuclein (αS). Interactions between αS and various cellular membranes are thought to be important to its native function as well as relevant to its role in disease. We use fluorescence correlation spectroscopy to investigate binding of αS to lipid vesicles as a function of the lipid composition and membrane curvature. We determine how these parameters affect the molar partition coefficient of αS, providing a quantitative measure of the binding energy, and calculate the number of lipids required to bind a single protein. Specific anionic lipids have a large effect on the free energy of binding. Lipid chain saturation influences the binding interaction to a lesser extent, with larger partition coefficients measured for gel-phase vesicles than for fluid-phase vesicles, even in the absence of anionic lipid components. Although we observe variability in the binding of the mutant proteins, differences in the free energies of partitioning are less dramatic than with varied lipid compositions. Vesicle curvature has a strong effect on the binding affinity, with a >15-fold increase in affinity for small unilamellar vesicles over large unilamellar vesicles, suggesting that αS may be a curvature-sensing protein. Our findings provide insight into how physical properties of the membrane may modulate interactions of αS with cellular membranes.     

Interaction between the NS4B amphipathic helix,AH2, and charged lipid headgroups alters membrane morphology and AH2 oligomeric state — Implications for the Hepatitis C virus life cycle

The non-structural protein 4B (NS4B) from Hepatitis C virus (HCV) plays a pivotal role in the remodelling of the host cell's membranes, required for the formation of the viral replication complex where genome synthesis occurs. NS4B is an integral membrane protein that possesses a number of domains vital for viral replication. Structural and biophysical studies have revealed that one of these, the second amphipathic N-terminal helix (AH2), plays a key role in these remodelling events. However, there is still limited understanding of the mechanism through which AH2 promotes these changes. Here we report on solid-state NMR and molecular dynamics studies that demonstrate that AH2 promotes the clustering of negatively charged lipids within the bilayer, a process that reduces the strain within the bilayer facilitating the remodelling of the lipid bilayer. Furthermore, the presence of negatively charged lipids within the bilayer appears to promote the disassociation of AH2 oligomers, highlighting a potential role for lipid recruitment in regulating NS protein interactions.     

Polymyxin binding to charged lipid membranes an example of cooperative lipid-protein interaction

The binding of polymyxin-B to lipid bilayer vesicles of synthesis phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescene probes to study the lipid phase transition.The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (with ΔT = 5°C) are observed. The upper transition (at T_u) is that of the pure lipid and the lower transition (at T₁) concerns the lipids bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1:1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin.With both fluorescence methods the fraction of lipid bound to polymxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction action of Ca²⁺ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca²⁺, polymyxin) interaction.A model is proposed which explains the association of polymyxing within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Salt stress alters membrane lipid content and lipid biosynthesis pathways in the plasma membrane and tonoplast

Plant cell membranes are the sites of sensing and initiation of rapid responses to changing environmental factors including salinity stress. Understanding the mechanisms involved in membrane remodeling is important for studying salt tolerance in plants. This task remains challenging in complex tissue due to suboptimal subcellular membrane isolation techniques. Here, we capitalized on the use of a surface charge-based separation method, free flow electrophoresis, to isolate the tonoplast (TP) and plasma membrane (PM) from leaf tissue of the halophyte ice plant (Mesembryanthemum crystallinum L.). Results demonstrated a membrane-specific lipidomic remodeling in this plant under salt conditions, including an increased proportion of bilayer forming lipid phosphatidylcholine in the TP and an increase in nonbilayer forming and negatively charged lipids (phosphatidylethanolamine and phosphatidylserine) in the PM. Quantitative proteomics showed salt-induced changes in proteins involved in fatty acid synthesis and desaturation, glycerolipid, and sterol synthesis, as well as proteins involved in lipid signaling, binding, and trafficking. These results reveal an essential plant mechanism for membrane homeostasis wherein lipidome remodeling in response to salt stress contributes to maintaining the physiological function of individual subcellular compartments.

Charge-based membrane fractionation techniques and tandem mass spectrometry combined with proteomic and lipidomic approaches reveal membrane-specific lipid remodeling in plants during salt stress.     

Phospholipid membranes form specific nonbilayer molecular arrangements that are antigenic.

Hexagonal phase (H(II))-preferring lipids such as phosphatidate, cardiolipin, and phosphatidylserine form nonbilayer molecular arrangements in lipid bilayers. While their presence in biological membranes has not been established, in vitro studies suggest that alterations in membrane properties modify their function. In this study, antiphospholipid monoclonal antibodies were developed against nonbilayer structures. One of the monoclonal antibodies identifies nonplanar surfaces in liposomes and in membranes of cultured cells. These results are the first evidence that natural membranes maintain a fragile balance between bilayer and nonbilayer lipid arrangements. Therefore, these antibodies can be used to evaluate the role of H(II)-preferring lipids in the modulation of membrane activities. Our studies demonstrated that nonplanar surfaces are highly immunogenic. Although these structures are normally transient, their formation can be stabilized by temperature variations, drugs, antibiotics, apolar peptides, and divalent cations. Our studies demonstrated that abnormal exposure of nonbilayer arrangements may induce autoimmune responses as found in the antiphospholipid syndrome.     

Manipulating the folding of membrane proteins: using the bilayer to our advantage

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.