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The use of colloid supported lipid bilayers (CSLBs) has recently been extended to create colloidal joints, that enable the assembly of structures with internal degrees of flexibility, and to study lipid membranes on curved and closed geometries. These novel applications of CSLBs rely on previously unappreciated properties: the simultaneous fluidity of the bilayer, lateral mobility of inserted (linker) molecules and colloidal stability. Here we characterize every step in the manufacturing of CSLBs in view of these requirements using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Specifically, we have studied the influence of different particle properties (roughness, surface charge, chemical composition, polymer coating) on the quality and mobility of the supported bilayer. We find that the insertion of lipopolymers in the bilayer can affect its homogeneity and fluidity. We improve the colloidal stability by inserting lipopolymers or double-stranded inert DNA into the bilayer. We include surface-mobile DNA linkers and use FRAP to characterize their lateral mobility both in their freely diffusive and bonded state. Finally, we demonstrate the self-assembly of flexibly linked structures from the CSLBs modified with surface-mobile DNA linkers. Our work offers a collection of experimental tools for working with CSLBs in applications ranging from controlled bottom-up self-assembly to model membrane studies.
Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides.
The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.
The apparent area expansion modulus and tensile strength of egg phosphatidylcholine (EPC) membranes are measured in the presence of monooleoylphosphatidylcholine (MOPC). The apparent area expansion modulus decreases from 171 mN m-1 for pure EPC membrane to 82 mN m-1 for a membrane containing 30 mol % MOPC. This significant decrease of the apparent area expansion modulus is attributed to the change of the membrane area due to the tension-dependent exchange of MOPC between the bathing solution and the membrane. Similar to the apparent area expansion modulus, the tensile strength of the membrane decreases with the increase of the molar concentration of MOPC in the membrane. The tensile strength of pure EPC membrane is 9.4 mN m-1 whereas that for a membrane containing 30 mol % MOPC is only 1.8 mN m-1, and for a membrane containing 50 mol % MOPC it is even smaller, on the order of 0.07 mN m-1. The decrease of the tensile strength is coupled with a decrease of the work for membrane breakdown, which changes from 4.3 x 10(-2) kT for pure EPC membrane to 2 x 10(-6) kT for a membrane with 50 mol % MOPC. Overall, these results show that the decrease of the apparent area expansion modulus in the presence of exchangeable molecules is a fundamental property for all membranes and depends on the area occupied by these molecules. The method presented here provides a unique tool for measuring the area occupied by an exchangeable molecule in the bilayer membrane.
Biomembrane interfaces create regions of slowed water dynamics in their vicinity. When two lipid bilayers come together, this effect is further accentuated, and the associated slowdown can affect the dynamics of larger-scale processes such as membrane fusion. We have used molecular dynamics simulations to examine how lipid and water dynamics are affected as two lipid bilayers approach each other. These two interacting fluid systems, lipid and water, both slow and become coupled when the lipid membranes are separated by a thin water layer. We show in particular that the water dynamics become glassy, and diffusion of lipids in the apposed leaflets becomes coupled across the water layer, while the "outer" leaflets remain unaffected. This dynamic coupling between bilayers appears mediated by lipid-water-lipid hydrogen bonding, as it occurs at bilayer separations where water-lipid hydrogen bonds become more common than water-water hydrogen bonds. We further show that such coupling occurs in simulations of vesicle-vesicle fusion prior to the fusion event itself. Such altered dynamics at membrane-membrane interfaces may both stabilize the interfacial contact and slow fusion stalk formation within the interface region.
A remarkable flexibility is observed in biological membranes, which allows them to form the structures of different curvatures. We addressed the question of intrinsic ability of phospholipid membranes to form highly curved structures and the role of cholesterol in this process. The distribution of cholesterol in the highly curved asymmetric DOPC/DOPS lipid bilayer was investigated by the coarse-grained molecular dynamics simulations in the membrane patches with large aspect ratio. It is shown that cholesterol induces uneven membrane curvature promoting the formation of extended flattened regions of the membrane interleaved by sharp bends. It is shown that the affinity of cholesterol to anionic DOPS or neutral DOPC lipids is curvature dependent. The cholesterol prefers DOPS to DOPC in either planar or highly curved parts of the membrane. In contrast, in the narrow interval of moderate membrane curvatures this preference is inverted. Our data suggest that there is a complex self-consistent interplay between the membrane curvature and cholesterol distribution in the asymmetric lipid bilayers. The suggested new function of cholesterol may have a biological relevance.
We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0-50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol's flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.
β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H-1H and 13C-13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6-8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.
Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and kainate type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively charged lipid bilayers in a phosphorylation-dependent manner and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.
It is crucial for molecular dynamics simulations of biomembranes that the force field parameters give a realistic model of the membrane behavior. In this study, we examined the OPLS3e force field for the carbon-hydrogen order parameters SCH of POPC (1-palmitoyl-2-oleoylphosphatidylcholine) lipid bilayers at varying hydration conditions and ion concentrations. The results show that OPLS3e behaves similarly to the CHARMM36 force field and relatively accurately follows the experimentally measured SCH for the lipid headgroup, the glycerol backbone, and the acyl tails. Thus, OPLS3e is a good choice for POPC bilayer simulations under many biologically relevant conditions. The exception are systems with an abundancy of ions, as similarly to most other force fields OPLS3e strongly overestimates the membrane-binding of cations, especially Ca2+. This leads to undesirable positive charge of the membrane surface and drastically lowers the concentration of Ca2+ in the surrounding solvent, which might cause issues in systems sensitive to correct charge distribution profiles across the membrane.
Flip-flop of lipids of the lipid bilayer (LBL) constituting the plasma membrane (PM) plays a crucial role in a myriad of events ranging from cellular signaling and regulation of cell shapes to cell homeostasis, membrane asymmetry, phagocytosis, and cell apoptosis. While extensive research has been conducted to probe the lipid flip flop of planar lipid bilayers (LBLs), less is known regarding lipid flip-flop for highly curved, nanoscopic LBL systems despite the vast importance of membrane curvature in defining the morphology of cells and organelles and in maintaining a variety of cellular functions, enabling trafficking, and recruiting and localizing shape-responsive proteins. In this paper, we conduct molecular dynamics (MD) simulations to study the energetics, structure, and configuration of a lipid molecule undergoing flip-flop and desorption in a highly curved LBL, represented as a nanoparticle-supported lipid bilayer (NPSLBL) system. We compare our findings against those of a planar substrate supported lipid bilayer (PSSLBL). Our MD simulation results reveal that despite the vast differences in the curvature and other curvature-dictated properties (e.g., lipid packing fraction, difference in the number of lipids between inner and outer leaflets, etc.) between the NPSLBL and the PSSLBL, the energetics of lipid flip-flop and lipid desorption as well as the configuration of the lipid molecule undergoing lipid flip-flop are very similar for the NPSLBL and the PSSLBL. In other words, our results establish that the curvature of the LBL plays an insignificant role in lipid flip-flop and desorption.
Cholesterol trafficking, which is an essential function in mammalian cells, is intimately connected to molecular-scale interactions through cholesterol modulation of membrane structure and dynamics and interaction with membrane receptors. Since these effects of cholesterol occur on micro- to millisecond timescales, it is essential to develop accurate coarse-grained simulation models that can reach these timescales. Cholesterol has been shown experimentally to thicken the membrane and increase phospholipid tail order between 0-40% cholesterol, above which these effects plateau or slightly decrease. Here, we showed that the published MARTINI coarse-grained force-field for phospholipid (POPC) and cholesterol fails to capture these effects. Using reference atomistic simulations, we systematically modified POPC and cholesterol bonded parameters in MARTINI to improve its performance. We showed that the corrections to pseudo-bond angles between glycerol and the lipid tails and around the oleoyl double bond particle (the "angle-corrected model") slightly improves the agreement of MARTINI with experimentally measured thermal, elastic, and dynamic properties of POPC membranes. The angle-corrected model improves prediction of the thickening and ordering effects up to 40% cholesterol but overestimates these effects at higher cholesterol concentration. In accordance with prior work that showed the cholesterol rough face methyl groups are important for limiting cholesterol self-association, we revised the coarse-grained representation of these methyl groups to better match cholesterol-cholesterol radial distribution functions from atomistic simulations. In addition, by using a finer-grained representation of the branched cholesterol tail than MARTINI, we improved predictions of lipid tail order and bilayer thickness across a wide range of concentrations. Finally, transferability testing shows that a model incorporating our revised parameters into DOPC outperforms other CG models in a DOPC/cholesterol simulation series, which further argues for its efficacy and generalizability. These results argue for the importance of systematic optimization for coarse-graining biologically important molecules like cholesterol with complicated molecular structure.
Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic α-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.
Increasing evidence suggests that the interaction of human islet amyloid polypeptide (hIAPP) with lipids may facilitate hIAPP aggregation and cause the death of pancreatic islet β-cells. However, the detailed hIAPP-membrane interactions and the influences of lipid compositions are unclear. In this study, as a first step to understand the mechanism of membrane-mediated hIAPP aggregation, we investigate the binding behaviors of hIAPP monomer at zwitterionic palmitoyloleoyl-phosphatidylcholine (POPC) bilayer by performing atomistic molecular dynamics simulations. The results are compared with those of hIAPP at anionic palmitoyloleoyl-phosphatidylglycerol (POPG) bilayers. We find that the adsorption of hIAPP to POPC bilayer is mainly initiated from the C-terminal region and the peptide adopts a helical structure with multiple binding orientations, while the adsorption to POPG bilayer is mostly initiated from the N-terminal region and hIAPP displays one preferential binding orientation, with its hydrophobic residues exposed to water. hIAPP monomer inserts into POPC lipid bilayers more readily than into POPG bilayers. Peptide-lipid interaction analyses show that the different binding features of hIAPP at POPC and POPG bilayers are attributed to different magnitudes of electrostatic and hydrogen-bonding interactions with lipids. This study provides mechanistic insights into the different interaction behaviors of hIAPP with zwitterionic and anionic lipid bilayers.
Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing "soft bilayers" with physiological levels of mechanical resistance (Young's modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.
Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D(lat) = 10-25 mum(2)/s and rotational diffusion coefficients ranging from D(rot) = 2.8 - 1.4 x 10(7) s(-1) were measured in pure liquid-disordered (L(d)) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L(o)) domains segregated from the L(d) phase at 23 degrees C. The lateral mobility of lipids in L(o) domains was around eightfold lower compared to those in the L(d) phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L(o) phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L(o) domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L(d) phase.
Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p-PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers.
In vitro reconstitutions of lipid membranes have proven to be an indispensable tool to rationalize their molecular complexity and to understand their role in countless cellular processes. However, amongst the various techniques used to reconstitute lipid bilayers in vitro, several approaches are not solvent-free, but rather contain residual hydrophobic solvents in between the two bilayer leaflets, generally as a consequence of the procedure used to generate the bilayer. To what extent the presence of these hydrophobic solvents modifies bilayer properties with respect to native, solvent-free, conditions remains an open question that has important implications for the appropriate interpretation of numerous experimental observations. Here, we thorouhgly characterize hydrophobic solvent-rich lipid bilayers using atomistic molecular dynamics simulations. Our data indicate that while the presence of hydrophobic solvents at high concentrations, such as hexadecane, has a significant effect on membrane thickness, their effects on surface properties, membrane order and lateral stress are quite moderate. Our results corroborate the validity of in vitro approaches as model systems for the investigations of biological membranes but raise a few cautionary aspects that must be considered when investigating specific membrane properties.
Inspired by the natural phospholipid structures for cell membrane, a synthetic phospholipid LC with an ion recognition group benzo-18-crown-6 (B18C6) moiety was prepared which has been demonstrated to be able to transport ions across the lipid bilayers. Fluorescent vesicle assay shows that LC has an excellent transport activity, and the EC50 value for K+ is 11.2 μM. The voltage clamp measurement exhibits regular square-like current signals with considerably long opening times, which indicates that LC achieves efficient ion transport through a channel mechanism and its single channel conductivity is 17 pS. Both of the vesicle assay and patch clamp tests indicate that LC has selectivity for Rb+, whose ionic radius is larger than the cavity of crown ether. It suggests that the sandwich interaction may play a key role in the ion transport across lipid bilayers. All these results help us to speculate that LC transports ions via a channel mechanism with a tetrameric aggregate as the active structure. In addition, LC had obvious toxicity to HeLa cells, and the IC50 was 100.0 μM after coculture for 36 h. We hope that this simple synthetic phospholipid will offer novel perspectives in the development of more efficient and selective ion transporters.
Phospholipids are a diverse group of biomolecules consisting of a hydrophilic headgroup and two hydrophobic acyl tails. The nature of the head and length and saturation of the acyl tails are important for defining the biophysical properties of lipid bilayers. It has recently been shown that the membranes of certain yeast species contain high levels of unusual asymmetric phospholipids consisting of one long and one medium-chain acyl moiety, a configuration not common in mammalian cells or other well-studied model yeast species. This raises the possibility that structurally asymmetric glycerophospholipids impart distinctive biophysical properties to the yeast membranes. Previously, it has been shown that lipids with asymmetric length tails form a mixed interdigitated gel phase and exhibit unusual endotherm behavior upon heating and cooling. Here, however, we address physiologically relevant temperature conditions and, using atomistic molecular dynamics simulations and environmentally sensitive fluorescent membrane probes, characterize key biophysical parameters (such as lipid packing, diffusion coefficient, membrane thickness, and area per lipid) in membranes composed of both length-asymmetric glycerophospholipids and ergosterol. Interestingly, we show that saturated but asymmetric glycerophospholipids maintain membrane lipid order across a wide range of temperatures. We also show that these asymmetric lipids can substiture of unsaturated symmetric lipids in the phase behaviour of ternary lipid bilayers. This may allow cells to maintain membrane fluidity, even in environments that lack oxygen, which is required for the synthesis of unsaturated lipids and sterols.
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