Light-activated control of protein channel assembly mediated by membrane mechanics.

Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

Structural studies of the lamellar to bicontinuous gyroid cubic (Q(G)(II)) phase transitions under limited hydration conditions.

Non-equilibrium pathways of lyotropic phase transitions such as the lamellar to inverse bicontinuous cubic phase transition are important dynamical processes resembling cellular fusion and fission processes which can be exploited in biotechnological processes such as drug delivery. However, utilising and optimising these structural transformations for applications require a detailed understanding of the energetic pathways which drive the phase transition. We have used the high pressure X-ray diffraction technique to probe the lamellar to Q(G)(II) phase transition in limited hydration monolinolein on the millisecond time scale. Our results show that the phase transition goes via a structural intermediate and once the Q(G)(II) phase initially forms the elastic energy in the bilayer drives this structure to its equilibrium lattice parameter.

Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.

The effects of pressure and temperature on the energetics and pivotal surface in a monoacylglycerol/water gyroid inverse bicontinuous cubic phase.

We have studied the effect of pressure and temperature on the location of the pivotal surface in a lipid inverse bicontinuous gyroid cubic phase (Q(G)(II)), described by the area at the pivotal surface (An), the volume between the pivotal surface and the bilayer midplane (Vn), and the molecular volume of the lipid (V). Small angle X-ray scattering (SAXS) was used to measure the swelling behaviour of the lipid, monolinolein, as a function of pressure and temperature, and the data were fitted to two different geometric models: the parallel interface model (PIM), and the constant mean curvature model (CMCM). The results show that an increase in temperature leads to a shift in the location of the pivotal surface towards the bilayer midplane, whilst an increase in pressure causes the pivotal surface to move towards the interfacial region. In addition, we describe the relevance of An, Vn and V for modeling the energetics of curved mesophases with specific reference to the mean curvature at the pivotal surface and discuss the significance of this parameter for modelling the energetics of curved mesophases.

Engineering de novo membrane-mediated protein-protein communication networks.

Mechanical properties of biological membranes are known to regulate membrane protein function. Despite this, current models of protein communication typically feature only direct protein-protein or protein-small molecule interactions. Here we show for the first time that, by harnessing nanoscale mechanical energy within biological membranes, it is possible to promote controlled communication between proteins. By coupling lipid-protein modules and matching their response to the mechanical properties of the membrane, we have shown that the action of phospholipase A(2) on acyl-based phospholipids triggers the opening of the mechanosensitive channel, MscL, by generating membrane asymmetry. Our findings confirm that the global physical properties of biological membranes can act as information pathways between proteins, a novel mechanism of membrane-mediated protein-protein communication that has important implications for (i) the underlying structure of signaling pathways, (ii) our understanding of in vivo communication networks, and (iii) the generation of building blocks for artificial protein networks.

Hydrostatic pressure effects on the lamellar to gyroid cubic phase transition of monolinolein at limited hydration.

Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.

Drug interactions with lipid membranes.

The field of drug-membrane interactions is one that spans a wide range of scientific disciplines, from synthetic chemistry, through biophysics to pharmacology. Cell membranes are complex dynamic systems whose structures can be affected by drug molecules and in turn can affect the pharmacological properties of the drugs being administered. In this tutorial review we aim to provide a guide for those new to the area of drug-membrane interactions and present an introduction to areas of this topic which need to be considered. We address the lipid composition and structure of the cell membrane and comment on the physical forces present in the membrane which may impact on drug interactions. We outline methods by which drugs may cross or bind to this membrane, including the well understood passive and active transport pathways. We present a range of techniques which may be used to study the interactions of drugs with membranes both in vitro and in vivo and discuss the advantages and disadvantages of these techniques and highlight new methods being developed to further this field.

Buffers may adversely affect model lipid membranes: a cautionary tale.

The effects of biological buffers on lipids have not been fully investigated because of the long-standing assumption that these buffers are too hydrophilic to substantially interact with the lipid membrane. We present evidence that for some buffers, this is not necessarily the case. Our research points toward a membrane softening effect caused by the buffer molecules interacting with the headgroup region of the lipid. Changes in the elastic properties of the membrane are known to control membrane protein behavior; this work serves as a warning for the design of assays utilizing model membranes in the presence of buffers.

A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs).

We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

A 3-D hexagonal inverse micellar lyotropic phase.

Lipids that are found in cell membranes form a variety of self-assembled phases in the presence of water. Many of these structures are liquid-crystalline with structural motifs mirrored in cells and organelles and can be exploited in the delivery of drugs and genes. We report the discovery of a lyotropic liquid crystalline phase based on a 3-D hexagonal close-packed arrangement of inverse micelles, of space group P6(3)/mmc. This is the first new inverse lyotropic liquid-crystalline phase to be reported for two decades and is the only known lyotropic phase whose structure consists of a close packing of identical inverse micelles.

Factors controlling the stability of a kinetically hindered lamellar-lamellar transition.

We show that we can manipulate the stability of a metastable gel phase, either to enhance its transitory nature or to "lock" it in. Using simple additives such as salt and fatty alcohol we were able to examine both the long-range effect, acting between charged bilayers, and short-range effects on the metastability. We found that the addition of salt to the cationic surfactant diethanolamine ester dimethyl ammonium chloride destabilized the gel phase, and at high concentrations it was able to decrease the length of time taken for the gel phase to revert to a hydrated solid "coagel" phase by an order of magnitude. The growth of the coagel phase was also found to be affected by increasing salt concentration, changing from needle-like (1D) to spherical growth. In contrast to the marked destabilization of the gel phase by salt, the addition of 1-octadecanol was found to prolong the lifetime of the gel phase almost indefinitely by disrupting the short-range packing between the surfactant molecules. This suggests that counterion binding plays a major role in the stability of metastable lamellar gel phases.

Spatial localization of PtdInsP2 in phase-separated giant unilamellar vesicles with a fluorescent PLC-delta 1 PH domain.

This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis.

Biophysical regulation of lipid biosynthesis in the plasma membrane.

We present a cellular model of lipid biosynthesis in the plasma membrane that couples biochemical and biophysical features of the enzymatic network of the cell-wall-less Mycoplasma Acholeplasma laidlawii. In particular, we formulate how the stored elastic energy of the lipid bilayer can modify the activity of curvature-sensitive enzymes through the binding of amphipathic alpha-helices. As the binding depends on lipid composition, this results in a biophysical feedback mechanism for the regulation of the stored elastic energy. The model shows that the presence of feedback increases the robustness of the steady state of the system, in the sense that biologically inviable nonbilayer states are less likely. We also show that the biophysical and biochemical features of the network have implications as to which enzymes are most efficient at implementing the regulation. The network imposes restrictions on the steady-state balance between bilayer and nonbilayer lipids and on the concentrations of particular lipids. Finally, we consider the influence of the length of the amphipathic alpha-helix on the efficacy of the feedback and propose experimental measurements and extensions of the modeling framework.

X-ray diffraction measurement of the monolayer spontaneous curvature of dioleoylphosphatidylglycerol.

Phosphatidylglycerol (PG) is an anionic lipid commonly found in large proportions in the cell membranes of bacteria and plants and, to a lesser extent, in animal cells. PG plays an important role in the regulation and determination of the elastic properties of the membrane. Using small angle X-ray scattering experiments, we obtain that the monolayer spontaneous curvature of dioleoylphosphatidylglycerol (DOPG) is -1/150+/-0.021 nm(-1) when measured in 150 mM NaCl. When the experiments are carried out in 150 mM NaCl and 20mM MgCl(2), the value obtained for the monolayer spontaneous curvature is -1/8.7+/-0.037 nm(-1). These values are of importance in modelling the effects of curvature elastic stress in membrane lipid homeostasis in the bacterium Acholeplasma laidlawii [Alley, S.H., Barahona, M., Ces, O., Templer, R.H., in press. Biophysical regulation of lipid biosynthesis in the plasma membrane. Biophys. J.] and indicate that divalent cations can play a significant role in altering curvature elastic stress.

New frontiers in single-cell analysis.

For this special issue of J. R. Soc. Interface we present an overview of the driving forces behind technological advances in the field of single-cell analysis. These range from increasing our understanding of cellular heterogeneity through to the study of rare cells, areas of research that cannot be tackled effectively using current high-throughput population-based averaging techniques.

Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool.

We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5-5 microm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles.

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.

Controlling the folding efficiency of an integral membrane protein.

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.

Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.

Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid-protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.