Orientational polarisability of lipid membrane surfaces

Here we present a fluorescence method based on the Stokes shift of the voltage-sensitive dye di-8-ANEPPS to quantify the orientational polarisability of lipid membrane surfaces, i.e. the polarisability due to molecular reorientation. Di-8-ANEPPS is already an established probe of membrane dipole potential. Its use, therefore, as a probe of both the dipole potential and orientational polarisability allows a direct comparison of these two properties in an identical region of the lipid bilayer. We applied the new technique on phosphatidylcholine vesicles to study the effects of different degrees of hydrocarbon saturation and of the incorporation of cholesterol and some of its oxidized derivatives. We found that lipids with unsaturated chains had a lower orientational polarisability than those with saturated chains. This could be explained by a reduction in membrane dipole potential as a result of a decrease in lipid packing density. Cholesterol derivatives were found to either increase or decrease the orientational polarisability depending on their molecular structure. The varying effects could be explained by antagonistic effects of the dipole potential and membrane order, which are both changed to varying degrees by the cholesterol derivatives and which lead to increases and decreases in orientational polarisability, respectively.     

Electrostatic interactions at charged lipid membranes. Hydrogen bonds in lipid membrane surfaces

Hydrogen-bonded structures within lipid membrane surfaces are not disrupted by water and are of thermodynamic and therefore potential structural importance in biological systems.     

Control of a redox reaction on lipid bilayer surfaces by membrane dipole potential

Nitro-2,1,3-benzoxadiazol-4-yl (NBD) group is a widely used, environment-sensitive fluorescent probe. The negatively charged dithionite rapidly reduces the accessible NBD-labeled lipids in liposomes to their corresponding nonfluorescent derivatives. In this study both the phospholipid headgroup and acyl chain NBD-labeled L-alpha-1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), respectively, were employed. The correlation of both the rate coefficient k(1) of the redox reaction and the fluorescence properties of the two probes with the membrane dipole potential Psi in fluid dipalmitoylglycerophosphocholine (DPPC) liposomes is demonstrated. When Psi of the bilayer was varied (decreased by phloretin or increased by 6-ketocholestanol), the value for k1 decreased for both DPPN and NBD-PC with increasing Psi. For both fluorophores a positive correlation to Psi was evident for the relative fluorescence emission intensity (RFI, normalized to the emission of the fluorophore in a DPPC matrix). The relative changes in emission intensity as a function of Psi were approximately equal for both NBD derivatives. Changes similar to those caused by phloretin were seen when dihexadecylglycerophosphocholine (DHPC) was added to DPPC liposomes, in keeping with the lower dipole potential for the former lipid compound compared with DPPC. These effects of Psi on NBD fluorescence should be taken into account when interpreting data acquired using NBD-labeled lipids as fluorescent probes.     

Penetration of lipid chains into transmembrane surfaces of membrane proteins: studies with MscL

The transmembrane surface of a multi-helix membrane protein will be rough with cavities of various sizes between the transmembrane alpha-helices. Efficient solvation of the surface by the lipid molecules that surround the protein in a membrane requires that the lipid fatty acyl chains be able to enter the cavities. This possibility has been investigated using fluorescence quenching methods. Trp residues have been introduced into lipid-facing sites in the first transmembrane alpha-helix (M1) of the mechanosensitive channel of large-conductance MscL; lipid-facing residues at the N-terminal end of M1 are buried below the transmembrane surface of the protein. Fluorescence emission maxima for lipid-facing Trp residues in M1 vary with position in the bilayer comparably to those for Trp residues in the second transmembrane alpha-helix (M2) despite the fact that lipid-facing residues in M2 are on the surface of the protein. Fluorescence emission spectra for most Trp residues on the periplasmic sides of M1 and M2 fit well to a model proposing a trough-like variation of dielectric constant across the membrane, but the relationship between location and fluorescence emission maximum on the cytoplasmic side of the membrane is more complex. The fluorescence of Trp residues in M1 is quenched efficiently by phospholipids with bromine-containing fatty acyl chains, showing that the lipid chains must be able to enter the Trp-containing cavities on the surface of MscL, resulting in efficient solvation of the surface.     

The electrostatics of lipid surfaces

Charged lipids constitute a substantial fraction of all membrane lipids. Their charges vary in quantity and distribution within their headgroup regions. In long range interactions, their charges' value and electrostatic potential in the vicinity of the membrane surface can be approximated by the Guy-Chapman theory. This theory treats the interface as a charged structureless plain surrounded by uniform environments. However, if one considers intermolecular interactions, such assumptions need to be revised. The interface is in reality a thick region containing the residual charges of lipid headgroups. Their arrangement depends on the type of lipid present in the membrane. The variety of lipids and their biological functions suggests that charge distribution determines the extent and type of interaction with surface associated molecules. Numerous examples show that protein behavior at the lipid bilayer surface is determined by the type of lipid present, indicating protein specificity towards certain surface locations and local properties (determined by lipid composition) of a particular type. Such specificity is achieved by a combination of electrostatic, hydrophobic and enthropic effects. Comparing lipid biological activity, it can be stated that residual charge distribution is one of the factors of intermolecular recognition leading to the specific interaction of lipid molecules and selected proteins in various processes, particularly those involved with signal transduction pathways. Such specificity enables a variety of processes occurring simultaneously on the same membrane surface to function without cross-reaction interference.     

Membrane on a chip: a functional tethered lipid bilayer membrane on silicon oxide surfaces

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin.     

A Mycobacterium tuberculosis-derived lipid inhibits membrane fusion by modulating lipid membrane domains

Tuberculosis is an infectious and potentially fatal disease caused by the acid-fast bacillus Mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. Lipoarabinomannan (LAM), an integral component of the MTB cell wall, is released when MTBs are taken into phagosomes and has been reported to be involved in the inhibition of phago-lysosomal (P-L) fusion. However, the physical chemistry of the effects of LAM on lipid membrane structure relative to P-L fusion has not been studied. We produced membranes in vitro composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol to simulate phagosomal lipid membranes and quantified the effects of the addition of LAM to these membranes, using fluorescence resonance energy transfer assays and atomic force microscopy. We found that LAM inhibits vesicle fusion and markedly alters lipid membrane domain morphology and sphingomyelin-chollesterol/dioleoylphosphatidylcholine ratios. These data demonstrate that LAM induces a dramatic reorganization of lipid membranes in vitro and clarifies the role of LAM in the inhibition of P-L fusion and the survival of the MTB within the macrophage.     

Structure and activity of lipid membrane biosensor surfaces studied with atomic force microscopy and a resonant mirror.

Three variants of the liposome fusion (coalescence) method to produce supported lipid bilayers, containing the ganglioside GM1 on silicon nitride surfaces, were studied. The first procedure involved attachment and fusion of liposomes containing DMPC, GM1 and a small amount of biotinylated lipid (Biotin-LC-DPPE) to a streptavidin coated surface. Direct fusion of liposomes composed of a mixture of DPPC, DPPG, DPPE, GM1 and cholesterol to the surface were the second variant. The final method utilised the second type of liposomes, fused onto a streptavidin layer with a small amount of exposed hydrophobic tails. The methods produced similar lipid layers, but with different ways of attachment to the surface. The binding of cholera toxin B-subunit (CTB) towards these sensor surfaces was measured in a resonant mirror biosensor instrument and the activity and longer-term stability of the layers were examined. The prepared surfaces were also imaged by atomic force microscopy (AFM) in liquid to characterise the topography of the lipid layers. The binding efficiency of CTB towards these surfaces was discussed in terms of lipid fluidity and surface roughness.     

Interaction of membrane skeletal proteins with membrane lipid domain

The object of this paper is to review briefly the studies on the interaction of red blood cell membrane skeletal proteins and their non-erythroid analogues with lipids in model systems as well as in natural membranes. An important question to be addressed is the physiological significance and possible regulatory molecular mechanisms in which these interactions are engaged.     

Cracking the membrane lipid code

Why has nature acquired such a huge lipid repertoire? Although it would be theoretically possible to make a lipid bilayer fulfilling barrier functions with only one glycerophospholipid, there are diverse and numerous different lipid species. Lipids are heterogeneously distributed across the evolutionary tree with lipidomes evolving in parallel to organismal complexity. Moreover, lipids are different between organs and tissues and even within the same cell, different organelles have characteristic lipid signatures. At the molecular level, membranes are asymmetric and laterally heterogeneous. This lipid asymmetry at different scales indicates that these molecules may play very specific molecular functions in biology. Some of these roles have been recently uncovered: lipids have been shown to be essential in processes such as hypoxia and ferroptosis or in protein sorting and trafficking but many of them remain still unknown. In this review we will discuss the importance of understanding lipid diversity in biology across scales and we will share a toolbox with some of the emerging technologies that are helping us to uncover new lipid molecular functions in cell biology and, step by step, crack the membrane lipid code.     

Enhancement of phototransduction protein interactions by lipid surfaces

The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G(t)), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G(t) assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTPgammaS (guanosine 5'-O-(thiotriphosphate)) form of Galpha(t) (Galpha(t)-GTPgammaS), minimizing functional heterogeneity of transducin and allowing activation with nanomolar Galpha(t)-GTPgammaS. Quantification of PDE activation and of the amount of Galpha(t)-GTPgammaS bound to PDE indicated that G(t) activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for Galpha(t)-GTPgammaS on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.     

Neuronal membrane lipid asymmetry.

The outer surface of intact synaptosomes was covalently labelled with trinitrobenzenesulfonic acid prior to isolation of the synaptic plasma membrane. Analysis of the membrane lipid demonstrated an asymmetric distribution of phospholipids across the synaptosomal plasma membrane. In addition, the fatty acyl composition of phosphatidylethanolamine from this neuronal membrane fraction was also distributed asymmetrically. The data are consistent with a $\text{de novo}$ generation of phospholipid asymmetry independent of serum lipid exchange processes. This structural asymmetry may have important consequences for neurotransmission.     

Diffusion-mediated localization on membrane surfaces.

Using the model of a cell membrane of a spherical surface in which membrane components may diffuse, the rate of localization due to trapping under diffusion control has been estimated by computing an analytical expression for the mean trapping time including the possibilities of a trapping probability less than 1 and/or the establishment of an equilibrium at the trap boundary.     

Charting the surfaces of the purple membrane

The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.     

Influence of membrane components in the binding of proteins to membrane surfaces

We have quantified the enhancement of membrane binding of activated and deactivated Galpha(s) and Galpha(q) subunits, Gbetagamma subunits, and phospholipase Cbeta(2) by lipid rafts and by the presence of membrane-associated protein partners. Membrane binding studies show that lipid rafts do not affect the intrinsic membrane affinity of Galpha(q)(GDP) and Galpha(s)(GDP), supporting the idea that these proteins partition evenly between the domains. Visualization of lipid rafts on monolayers by use of a probe that does not enter raft domains shows that neither activated nor deactivated Galpha(q)(GDP) subunits distribute evenly between the raft and nonraft domains, contrary to previous suggestions. Membrane binding of deactivated Galpha(q) and Galpha(s)(GDP) became weaker when Gbetagamma subunits were present, in contrast with the behavior predicted by thermodynamics. However, activated Galpha subunits and phospholipase Cbeta(2) were recruited to membrane surfaces by protein partners by predicted amounts. Our studies suggest that the anomalous behavior seen for deactivated Galpha subunits in the presence of Gbetagamma subunits may be due to conformational changes in the N-terminus and/or occlusion of a portion of its membrane interaction region by Gbetagamma. Even though membrane recruitment was clearly observed for one protein partner, the presence of a second partner of lower affinity did not further promote membrane binding. For these proteins, the formation of larger protein complexes with very high membrane affinities is unlikely.     

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.