Supported Bilayers with Excess Membrane Reservoir: A Template for Reconstituting Membrane Budding and Fission

A complete mechanistic understanding of membrane-localized processes in vesicular transport, such as membrane budding and fission, requires their reconstitution with biochemically-defined components from a biochemically-defined substrate. Supported bilayers formed by vesicle fusion represent an attractive substrate for this purpose. However, conventional supported bilayers lack a sufficient membrane reservoir to recreate membrane budding and fission events. We describe the formation of supported bilayers with excess membrane reservoir (SUPER) templates from the fusion of liposomes containing negatively charged lipids on silica beads under high-ionic-strength conditions. Using a fluorescence microscopy-based assay to monitor early and late stages of supported bilayer formation, we show that an increase in ionic strength leads to an increase in the rates of liposome adsorption and subsequent fusion during formation of supported bilayers. The two rates, however, increase disproportionally, leading to accumulation of excess reservoir with an increase in ionic strength. SUPER templates allow the seamless application of microscopy-based assays to analyze membrane-localized processes together with sedimentation-based assays to isolate vesicular and nonvesicular products released from the membrane. The results presented here emphasize the general utility of these templates for analyzing vesicular and nonvesicular transport processes.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

Quantification of phase transitions of lipid mixtures from bilayer to non-bilayer structures: Model,experimental validation and implication on membrane fusion

Lipid bilayers provide a solute-proof barrier that is widely used in living systems. It has long been recognized that the structural changes of lipids during the phase transition from bilayer to non-bilayer have striking similarities with those accompanying membrane fusion processes. In spite of this resemblance, the numerous quantitative studies on pure lipid bilayers are difficult to apply to real membranes. One reason is that in living matter, instead of pure lipids, lipid mixtures are involved and there is currently no model that establishes the connection between pure lipids and lipid mixtures. Here, we make this connection by showing how to obtain (i) the short-range repulsion between bilayers made of lipid mixtures and, (ii) the pressure at which transition from bilayer phase to non-bilayer phases occur. We validated our models by fitting the experimental data of several lipid mixtures to the theoretical data calculated based on our model. These results provide a useful tool to quantitatively predict the behavior of complex membranes at low hydration.     

Supported double membranes

Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin–streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Vesicle diffusion close to a membrane: intermembrane interactions measured with fluorescence correlation spectroscopy

The protein machinery controlling membrane fusion (or fission) has been well studied; however, the role of vesicle diffusion near membranes in these critical processes remains unclear. We experimentally and theoretically investigated the dynamics of small vesicles (∼50 nm in diameter) that are diffusing near supported planar bilayers acting as “target” membranes. Using total internal reflection-fluorescence correlation spectroscopy, we examined the validity of theoretical analyses of vesicle-membrane interactions. Vesicles were hindered by hydrodynamic drag as a function of their proximity to the planar bilayer. The population distributions and diffusion kinetics of the vesicles were further affected by changing the ionic strength and pH of the buffer, as well as the lipid composition of the planar membrane. Effective surface charges on neutral bilayers were also analyzed by comparing experimental and theoretical data, and we show the possibility that vesicle dynamics can be modified by surface charge redistribution of the planar bilayer. Based on these results, we hypothesize that the dynamics of small vesicles, diffusing close to biomembranes, may be spatially restricted by altering local physiological conditions (e.g., salt concentration, lipid composition, and pH), which may represent an additional mechanism for controlling fusion (or fission) dynamics.     

Electrostimulated fusion and fission of bilayer lipid membranes

The interaction and fusion of black lipid membranes have been studied. Two planar bilayers were simultaneously inflated towards each other; when they made contact, spontaneous formation of a ‘trilaminar structure’ (one bilayer bounded by two bilayers along the perimeter) was observed. Application of a discrete voltage pulse gave rise to the formation of a cylindrical membrane, that is, to the fusion of two bilayers. It is shown that fusion results from electrical breakdown in the contact region of the ‘trilaminar structure’.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the ³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. ²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. ³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, ³¹P and ²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from −31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.     

Aggregation of model membrane proteins, modulated by hydrophobic mismatch, membrane curvature, and protein class

Aggregation of transmembrane proteins is important for many biological processes, such as protein sorting and cell signaling, and also for in vitro processes such as two-dimensional crystallization. We have used large-scale simulations to study the lateral organization and dynamics of lipid bilayers containing multiple inserted proteins. Using coarse-grained molecular dynamics simulations, we have studied model membranes comprising ∼7000 lipids and 16 identical copies of model cylindrical proteins of either α-helical or β-barrel types. Through variation of the lipid tail length and hence the degree of hydrophobic mismatch, our simulations display levels of protein aggregation ranging from negligible to extensive. The nature and extent of aggregation are shown to be influenced by membrane curvature and the shape or orientation of the protein. Interestingly, a model β-barrel protein aggregates to form one-dimensional strings within the bilayer plane, whereas a model α-helical bundle forms two-dimensional clusters. Overall, it is clear that the nature and extent of membrane protein aggregation is dependent on several aspects of the proteins and lipids, including hydrophobic mismatch, protein class and shape, and membrane curvature.     

Lipid bilayer deformation and the free energy of interaction of a Kv channel gating-modifier toxin

A number of membrane proteins act via binding at the water/lipid bilayer interface. An important example of such proteins is provided by the gating-modifier toxins that act on voltage-gated potassium (Kv) channels. They are thought to partition to the headgroup region of lipid bilayers, and so provide a good system for probing the nature of interactions of a protein with the water/bilayer interface. We used coarse-grained molecular dynamics simulations to compute the one-dimensional potential of mean force (i.e., free energy) profile that governs the interaction between a Kv channel gating-modifier toxin (VSTx1) and model phospholipid bilayers. The reaction coordinate sampled corresponds to the position of the toxin along the bilayer normal. The course-grained representation of the protein and lipids enabled us to explore extended time periods, revealing aspects of toxin/bilayer dynamics and energetics that would be difficult to observe on the timescales currently afforded by atomistic molecular dynamics simulations. In particular, we show for this model system that the bilayer deforms as it interacts with the toxin, and that such deformations perturb the free energy profile. Bilayer deformation therefore adds an additional layer of complexity to be addressed in investigations of membrane/protein systems. In particular, one should allow for local deformations that may arise due to the spatial array of charged and hydrophobic elements of an interfacially located membrane protein.     

Biomimetic membrane systems to study cellular organization

During many cellular processes such as cell division, polarization and motility, the plasma membrane does not only represent a passive physical barrier, but also provides a highly dynamic platform for the interplay between lipids, membrane binding proteins and cytoskeletal elements. Even though many regulators of these interactions are known, their mutual interdependence appears to be highly complex and difficult to study in a living cell. Over the past few years, in vitro studies on membrane–cytoskeleton interactions using biomimetic membranes turned out to be extremely helpful to get better mechanistic insight into the dynamics of these processes. In this review, we discuss some of the recent developments using in vitro assays to dissect the role of the players involved: lipids in the membrane, proteins binding to membranes and proteins binding to membrane proteins. We also summarize advantages and disadvantages of supported lipid bilayers as model membrane.     

Probing the Huntingtin 1-17 Membrane Anchor on a Phospholipid Bilayer by Using All-Atom Simulations

Mislocalization and aggregation of the huntingtin protein are related to Huntington’s disease. Its first exon—more specifically the first 17 amino acids (Htt17)—is crucial for the physiological and pathological functions of huntingtin. It regulates huntingtin’s activity through posttranslational modifications and serves as an anchor to membrane-containing organelles of the cell. Recently, structure and orientation of the Htt17 membrane anchor were determined using a combined solution and solid-state NMR approach. This prompted us to refine this model by investigating the dynamics and thermodynamics of this membrane anchor on a POPC bilayer using all-atom, explicit solvent molecular dynamics and Hamiltonian replica exchange. Our simulations are combined with various experimental measurements to generate a high-resolution atomistic model for the huntingtin Htt17 membrane anchor on a POPC bilayer. More precisely, we observe that the single α-helix structure is more stable in the phospholipid membrane than the NMR model obtained in the presence of dodecylphosphocholine detergent micelles. The resulting Htt17 monomer has its hydrophobic plane oriented parallel to the bilayer surface. Our results further unveil the key residues interacting with the membrane in terms of hydrogen bonds, salt-bridges, and nonpolar contributions. We also observe that Htt17 equilibrates at a well-defined insertion depth and that it perturbs the physical properties—order parameter, thickness, and area per lipid—of the bilayer in a manner that could favor its dimerization. Overall, our observations reinforce and refine the NMR measurements on the Htt17 membrane anchor segment of huntingtin that is of fundamental importance to its biological functions.     

Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs—cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)—on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx—the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate.     

Molecular dynamics simulation of lipid packing and mobility in bilayer membranes

A model of lipid bilayer membrane in water has been developed. Parameters have been selected that allow molecular dynamics simulation of lipid bilayers in the all-atom approximation. The calculated indices of packing and mobility of lipid molecules for the liquid crystalline state of the bilayer agree well with the experimental data. Based on the model of the liquid crystalline state of the membrane, a system in the gel-like state has been constructed. The gel-state model reproduces well the packing of lipids in real bilayers, whereas the mobility of molecules proves to be overestimated.     

Molecular simulation of rapid translocation of cholesterol, diacylglycerol, and ceramide in model raft and nonraft membranes

The translocation of lipids across membranes (flip-flop) is an important biological process. Slow exchange on a physiological timescale allows the creation of asymmetric distributions of lipids across cellular membranes. The location of lipids and their rate of exchange have important biological consequences, especially for lipids involved in cellular signaling. We investigated the translocation of cholesterol, ceramide, and diacylglycerol in two model bilayers using molecular dynamics simulations. We estimate half times for flip-flop for cholesterol, diacylglycerol, and ceramide of 20 μs, 30 μs, and 10 ms in a POPC bilayer, compared with approximately 30 min, 30 ms, and 30 s in a model raft bilayer (1:1:1 PSM, POPC, and cholesterol). Cholesterol has a large (54 kJ/mol) free energy of exchange between the POPC and raft bilayer, and therefore, it strongly prefers the more ordered and rigid raft bilayer over the more liquid POPC bilayer. Ceramide and diacylglycerol have relatively small free energies of exchange, suggesting nearly equal preference for both bilayers. This unexpected result may have implications for ceramide and diacylglycerol signaling and membrane localization.     

beta-Sheet structured beta-amyloid(1-40) perturbs phosphatidylcholine model membranes

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Abeta40 assemblies.     

How calcium may cause exocytosis in sea urchin eggs

The process of secretory granule-plasma membrane fusion can be studied in sea urchin eggs. Micromolar calcium concentrations are all that is required to bring about exocytosisin vitro. I discuss recent experiments with sea urchin eggs that concentrate on the biophysical aspects of granule-membrane fusion. The backbone of biological membranes is the lipid bilayer. Sea urchin egg membrane lipids have negatively charged head groups that give rise to an electrical potential at the bilayer-water interface. We have found that this surface potential can affect the calcium required for exocytosis. Effects on the surface potential may also explain why drugs like trifluoperazine and tetracaine inhibit exocytosis: they absorb to the bilayer and reduce the surface potential. The membrane lipids may also be crucial to the formation of the exocytotic pore through which the secretory granule contents are released. We have measured calcium-induced production of the lipid, diacylglycerol. This lipid can induce a phase transition that will promote fusion of apposed lipid bilayers. The process of exocytosis involves the secretory granule core as well as the lipids of the membrane. The osmotic properties of the granule contents lead to swelling of the granule during exocytosis. Swelling promotes the dispersal of the contents as they are extruded through the exocytotic pore. The movements of water and ions during exocytosis may also stabilize the transient fusion intermediate and consolidate the exocytotic pore as fusion occurs.     

Molecular dynamics simulations of the lipid bilayer edge

Phospholipid bilayers have been intensively studied by molecular dynamics (MD) simulation in recent years. The properties of bilayer edges are important in determining the structure and stability of pores formed in vesicles and biomembranes. In this work, we use molecular dynamics simulation to investigate the structure, dynamics, and line tension of the edges of bilayer ribbons composed of pure dimyristoylphosphatidylcholine (DMPC) or palmitoyl-oleoylphosphatidylethanolamine (POPE). As expected, we observe a significant reorganization of lipids at and near the edges. The treatment of electrostatic effects is shown to have a qualitative impact on the structure and stability of the edge, and significant differences are observed in the dynamics and structure of edges formed by DMPC and palmitoyl-oleoylphosphatidylethanolamine. From the pressure anisotropy in the simulation box, we calculate a line tension of approximately 10-30 pN for the DMPC edge, in qualitative agreement with experimental estimates for similar lipids.