Phosphoglycerol Research Articles

PFAS fluidize synthetic and bacterial lipid monolayers based on hydrophobicity and lipid charge

Poly- and Perfluoroalkyl substances (PFASs) are pollutants of emerging concern that persist in nature and pose environmental health and safety risks. PFAS disrupt biological membranes resulting in cellular inhibition, but the mechanism of disruption and the role of lipid composition remain unclear. We examine the role of phospholipid saturation and headgroup charge on the interactions between PFASs and phospholipid monolayers comprised of synthetic phosphocholine (PC) and phosphoglycerol (PG) lipids and prepared from bacteria membrane extracts rich in PG lipids from an environmentally relevant marine bacterium Alcanivorax borkumensis. When deposited on a buffered subphase containing PFAS, PFAS mixed within and fluidized zwitterionic and net-anionic monolayers leading to increases in monolayer compressibility that were driven by a combination of PFAS hydrophobicity and monolayer charge density. Differences in the monolayer response using saturated or unsaturated lipids are attributed to the ability of the unsaturated lipids to accommodate PFAS within ‘void space’ arising from the bent lipid tails. Similar fluidization and compressibility behavior were also observed in A. borkumensis lipid monolayers. This work provides new insight into PFAS partitioning into bacterial membranes and the effect PFAS have on the physicomechanical properties of zwitterionic and charged lipid monolayers.

Changes in the Distribution of Membrane Lipids during Growth of Thermotoga maritima at Different Temperatures: Indications for the Potential Mechanism of Biosynthesis of Ether-Bound Diabolic Acid (Membrane-Spanning) Lipids.

ABSTRACTMembrane-spanning lipids are present in a wide variety of archaea, but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C30, C32, and C34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C30 and C32 diabolic acids, i.e., dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but are preferably produced when the length of the acyl chains is not optimal. Compared with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) head groups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process is a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima that is also present in other bacteria producing sn-1-alkyl ether lipids but not plasmalogens, suggesting it is involved in the conversion of the ester-to-ether bond in the diabolic acids bound in membrane-spanning lipids.IMPORTANCE Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales. The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipid composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipid biosynthetic process, which can further clarify the pathway leading to the biosynthesis of these compounds.

Structural Organization of Cardiolipin-Containing Vesicles as Models of the Bacterial Cytoplasmic Membrane.

The bacterial cytoplasmic membrane is the innermost bacterial membrane and is mainly composed of three different phospholipid species, i.e., phosphoethanolamine (PE), phosphoglycerol (PG), and cardiolipin (CL). In particular, PG and CL are responsible for the negative charge of the membrane and are often the targets of cationic antimicrobial agents. The growing resistance of bacteria toward the available antibiotics requires the development of new and more efficient antibacterial drugs. In this context, studying the physicochemical properties of the bacterial cytoplasmic membrane is pivotal for understanding drug-membrane interactions at the molecular level as well as for designing drug-testing platforms. Here, we discuss the preparation and characterization of PE/PG/CL vesicle suspensions, which contain all of the main lipid components of the bacterial cytoplasmic membrane. The vesicle suspensions were characterized by means of small-angle neutron scattering, dynamic light scattering, and electron paramagnetic spectroscopy. By combining solution scattering and spectroscopy techniques, we propose a detailed description of the impact of different CL concentrations on the structure and dynamics of the PE/PG bilayer. CL induces the formation of thicker bilayers, which exhibit higher curvature and are overall more fluid. The experimental results contribute to shed light on the structure and dynamics of relevant model systems of the bacterial cytoplasmic membrane.

The Mechanisms of Lipid-Mediated Regulation of the Activity of Antimicrobial Peptides of Different Secondary Structure

The present study focuses on the membrane activity of antimicrobial peptides of different secondary structure: bactenecin, an antimicrobial peptide, which forms a loop stabilized by a single disulfide bond; defensins, antimicrobial peptides of the beta-sheet structure that are stabilized by three disulfide bonds; and pediocin, lantibiotic with a combined secondary structure, including both beta-sheet and alpha-helix regions. It has been found that bactenecin forms ion-permeable nanopores of various conductance in lipid bilayers composed of phosphocholine (PC) and phosphoglycerol (PG) independently on saturation of their hydrocarbon chains.

Bioinspired Nanoemulsions Stabilized by Phosphoethanolamine and Phosphoglycerol Lipids.

Water-in-oil (W/O) nanoemulsions stabilized by phospholipids (PLs) are increasingly exploited in a wide spectrum of applications, from pharmaceuticals to food and cosmetic formulations. In this work, we report the design and optimization of an innovative emulsion based on a mixture of phosphoethanolamine (PE) and phosphoglycerol (PG) PLs, inspired by the composition of the inner leaflet of a bacterial outer membrane. Using the natural oil squalene as the continuous organic phase, no additional emulsion stabilizer is needed. On the other hand, a small amount of Span 80 is required when dodecane is used. The obtained nanoemulsions are stable for at least two hours, thus allowing the droplet size and distribution to be characterized by Dynamic Light Scattering (DLS) and the lipid layer structure and dynamics to be analyzed by Electron Paramagnetic Resonance (EPR) spectroscopy. The results indicate that squalene shallowly intercalates among the lipid tail termini, being unable to deeply penetrate the adsorbed lipid monolayer. The altered lipid dynamics are proposed to be the reason for the enhanced emulsion stability, this paving the way to future implementations and possible applications.

Effects of Polyunsaturated Fatty Acids and Metallation on the Antimicrobial Activity and Membrane-Disruptive Properties of Host-Defense Metallopeptide PISCIDIN 1

Piscidins are host-defense peptides that exhibit properties critical for the development of novel therapeutics. While the capability of piscidins to physically disrupt membranes is well accepted, their ability to bind Cu2+ and chemically damage membranes is less understood. Metallation lowers their charge, which could affect membrane binding. It also provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Such mechanistic possibility is important to examine since bacteria such as Vibrio species incorporate exogenous poly-UFA (PUFA) in their membranes to affect membrane properties and virulence. Here, we investigated how metallation and the UFA-content of membranes influence the mechanism of action and antimicrobial potency of P1, a particularly membrane-active piscidin. First, using dye leakage experiments with model membranes constituted of phosphoglycerol (PG) and phosphatidylcholine (PC), we quantified the permeabilization of P1/P1-Cu on membranes containing 10% of either 18:2 PUFA or Aldo-PC, its oxidized version. We found that the combined effect of metallating P1 and making Aldo-PC available yielded a ten-fold increase in membranolysis. Second, we used 31P solid-state NMR to characterize the partitioning behaviors of P1/P1-Cu in these membranes. Both peptides demonstrated stronger affinity for PG than PC. This effect was more pronounced for P1-Cu than P1 in the Aldo-PC-containing bilayers. Hence, differences in the membrane partitioning of P1 and P1-Cu in OxPL-containing membranes underlie their contrasted permeabilization effects. Third, we probed how the changing PUFA-content of bacterial cell membranes affects virulence upon P1/P1-Cu exposure. When V. cholerae bacteria were fed specific PUFAs (18:2; 20:4; 20:5; 22:6) and either peptide, peptide activity was enhanced with all FAs, except the 20:4. This demonstrates the importance of considering exogenous UFAs in antimicrobial treatments.

Connections between Macroscopic Wettability and Lipid Structures on Clay Montmorillonite

Soil organic matter, which includes lipids from bacterial membranes, influences characteristics of soil such as soil water repellency. However, the mechanism that governs the distribution of lipids on mineral surfaces and the consequent alteration of macroscale wettability is unclear. This project investigates phospholipid-clay films to understand how lipids influence wettability. Previous investigations conducted to study the physical characteristics of the lipid-mineral surfaces focused on phosphoethanolamine (PE) lipids on the clay mineral montmorillonite. In this study, the physical characteristics of phosphoglycerol (PG) lipids on montmorillonite were studied. As PE lipids are zwitterionic and PG lipids are negatively charged, these lipids interact with the clay surface differently and can be used to determine how lipid-clay interaction alters wettability. The physical characteristics of the lipid-mineral films were studied using atomic force microscopy and fluorescence microscopy. Atomic force microscopy experiments were conducted to study the topography of the PG-montmorillonite surfaces while fluorescence microscopy was employed to map the distribution of the lipids on the montmorillonite surface. Additionally, wettability experiments were conducted on the surface of the PG-montmorillonite samples to determine the kinetics of water soaking into the film. Physical characteristics were then compared to wettability data to establish causative relationships. We find that the small-scale distribution of lipid aggregates has the most impact on macroscopic wettability for PG lipids, whereas, for PE lipids, the lipid distribution across hundreds of microns was the main influence.

Soluble Regions of GlpG Influence Protein-Lipid Interactions and Lipid Distribution.

The GlpG rhomboid protease from E. coli is a well-characterized intramembrane protease that cleaves transmembrane substrates inside the lipid bilayer. Most studies have focused on the GlpG transmembrane domain containing the catalytic site, while the full-length protein, also containing a soluble cytoplasmic domain, a linker region, and a small positively charged C-terminal fragment, remains poorly understood. In this work, we used coarse-grained molecular dynamics (CGMD) simulations to investigate full-length GlpG embedded in a native-like model of the E. coli membrane. We identified differences in the distribution and clustering of phosphoglycerol(PG)-based lipids around GlpG in both leaflets depending on whether the soluble regions are present or absent. These data suggest a possible role of the cytoplasmic extensions of GlpG in the regulation of the lipid environment around GlpG, which may influence the activity of GlpG in vivo.

Cu2+-Directed Liposome Membrane Fusion, Positive-Stain Electron Microscopy, and Oxidation.

Natural lipid headgroups contain a few types of metal ligands, such as phosphate, amine, and serine, which interact with metal ions differently. Herein, we studied the binding between Cu2+ and liposomes with four types of headgroups: phosphocholine (PC), phosphoglycerol (PG), phosphoserine (PS), and cholinephosphate (CP). Using fluorescently headgroup-labeled liposomes, Cu2+ strongly quenched the CP and PS liposomes, whereas quenching of PC and PG was weaker. Dynamic light scattering indicated that all of the four liposomes aggregated at high Cu2+ concentrations, and ethylenediaminetetraacetic acid (EDTA) only restored the original size of the PC liposome, implying fusion of the other three types of liposomes. The leakage tests revealed that the integrity of PC liposomes was not affected by Cu2+, but the other three liposomes leaked. Under TEM, all of the liposomes show a positive-stain feature in the presence of Cu2+ and Cu2+-stained individual liposomes with a short incubation time (<1 min). The oxidative catalytic property of Cu2+ was also tested, and a tight binding by the PS liposome inhibited the activity of Cu2+. Finally, a model of interaction for each liposome was proposed, and each one has a different metal-binding and interaction mechanism.

Hydrogen-Bond Networks near Supported Lipid Bilayers from Vibrational Sum Frequency Generation Experiments and Atomistic Simulations.

We report vibrational sum frequency generation (SFG) spectra in which the C-H stretches of lipid alkyl tails in fully hydrogenated single- and dual-component supported lipid bilayers are detected along with the O-H stretching continuum above the bilayer. As the salt concentration is increased from ∼10 μM to 0.1 M, the SFG intensities in the O-H stretching region decrease by a factor of 2, consistent with significant absorptive-dispersive mixing between χ(2) and χ(3) contributions to the SFG signal generation process from charged interfaces. A method for estimating the surface potential from the second-order spectral lineshapes (in the OH stretching region) is presented and discussed in the context of choosing truly zero-potential reference states. Aided by atomistic simulations, we find that the strength and orientation distribution of the hydrogen bonds over the purely zwitterionic bilayers are largely invariant between submicromolar and hundreds of millimolar concentrations. However, specific interactions between water molecules and lipid headgroups are observed upon replacing phosphocholine (PC) lipids with negatively charged phosphoglycerol (PG) lipids, which coincides with SFG signal intensity reductions in the 3100-3200 cm-1 frequency region. The atomistic simulations show that this outcome is consistent with a small, albeit statistically significant, decrease in the number of water molecules adjacent to both the lipid phosphate and choline moieties per unit area, supporting the SFG observations. Ultimately, the ability to probe hydrogen-bond networks over lipid bilayers holds the promise of opening paths for understanding, controlling, and predicting specific and nonspecific interactions between membranes and ions, small molecules, peptides, polycations, proteins, and coated and uncoated nanomaterials.

An extensive simulation study of lipid bilayer properties with different head groups, acyl chain lengths, and chain saturations

Previous MD simulations of six phosphocholine (PC) lipid bilayers demonstrated the accuracy of the CHARMM36 force field (C36FF) for PC bilayer simulation at varied temperatures (BBA-Biomembranes, 1838 (2014): 2520–2529). In this work, we further examine the accuracy of C36FF over a wide temperature range for a broader range of lipid types such as various head groups (phosphatidic acid (PA), PC, phosphoethanolamine (PE), phosphoglycerol (PG), and phosphoserine (PS)), and tails (saturated, mono-, mixed- and poly-unsaturated acyl chains with varied chain lengths). The structural properties (surface area per lipid (SA/lip), overall bilayer thickness, hydrophobic thickness, headgroup-to-headgroup thickness, deuterium order parameter (SCD), and spin-lattice relaxation time (T1)) obtained from simulations agree well with nearly all available experimental data. Our analyses indicate that PS lipids have the most inter-lipid hydrogen bonds, while PG lipids have the most intra-lipid hydrogen bonds, which play the main role in their low SA/lip in PS lipids and low thicknesses in PG lipids, respectively. PS, PE, and PA lipids have the largest contact clusters with on average 5–8 lipids per cluster, while PC and PG have clusters of 4 lipids based on a cutoff distance of 6.5Å. PS lipids have much slower lipid wobble (i.e., higher correlation time) than other head groups at a given temperature as the hydrogen bonded network significantly reduces a lipid's mobility, and the rate of lipid wobble increases dramatically as temperature increases. These in-depth analyses facilitate further understanding of lipid bilayers at the atomic level.

The Transmembrane State of Antimicrobial Piscidin in Bacterial Cell Membrane Mimics Dramatically Alters the Polar-Nonpolar Segregation of the Bilayer

Piscidin 1 (P1) and piscidin (P3) 3 are twenty-two-residue-long antimicrobial peptides that are membrane-active. While their amphipathic structures lend themselves to forming pores by the carpet/toroidal pore mechanism, direct evidence for their mode of action is lacking. Previously, we determined the high resolution structures of both peptides bound parallel to the surface of bacterial cell mimics constituted of 3:1 phosphocholine (PC)/phosphoglycerol (PG). In a comprehensive effort to decipher their mechanism of action, we combined dye leakage assays, oriented CD (OCD), X-ray diffraction (XRD), and neutron diffraction (ND) in 3:1 PC/PG to simultaneously investigate at various peptide-to-lipid (P/L) ratios the permeabilization capability of the peptides, the physical state of the bilayer, and the bilayer orientations and locations of the peptides.The OCD results show that P1 adopted an inserted state at a lower P/L than P3 (1:25 versus 1:16) in agreement with its stronger permeabilization capability. XRD data showed that the bilayer gradually thinned from 52.5 A in the peptide-free state to 48.5 A in the presence of piscidin at 1:16 P/L. ND experiments at 1:25 P/L confirmed the OCD and XRD results. Because we calculated the ND profiles for samples containing peptides that were 2H-labeled at either the C-end or at both the N- and C-ends, we could determine the bilayer orientation and location of each peptide. Importantly, the combined OCD and ND data revealed that P1 has a transmembrane orientation almost parallel to the bilayer normal. Further investigation of this state at a 1:16 P/L demonstrated that it induces a dramatic broadening of the lipid headgroup distribution indicating that it abolishes the strict nonpolar-polar segregation of the bilayer. The implications for piscidin's mode of action will be discussed.

Evaluating the role of conserved amino acids in bacterial O-oligosaccharyltransferases by in vivo, in vitro and limited proteolysis assays

Bacterial O-Oligosaccharyltransferases (O-OTases) constitute a growing family of enzymes that catalyze the transfer of a glycan from a lipid carrier to protein acceptors. O-OTases are inner membrane proteins that display limited sequence similarity, except for the Wzy_C signature domain also present in a predicted periplasmic loop of the WaaL ligase, the enzyme responsible for transferring the O antigen to the lipid A core. The mechanism of O-OTase-dependent glycosylation is poorly understood. In this work, conserved amino acid residues in the O-OTases were replaced with alanine in PglL, the O-OTase of Neisseria meningitidis. The activity of wild-type PglL and its mutant derivatives were analyzed in vivo in engineered Escherichia coli cells, and in in vitro assays. We identified two additional sites of pilin glycosylated exclusively by PglL in E. coli. Both sites are modified with phosphoglycerol (PG) by different enzymes in Neisseria gonorrhoeae and Neisseria meningitidis. Limited proteolysis experiments revealed a conformational change that is triggered upon interaction of the C-terminal region of PglL with the lipid-linked oligosaccharide (LLO) substrate. These experiments showed that Q178 and Y405 are required for optimal function, whereas H349 is essential for activity and plays a critical role in the interaction with LLO. The equivalent His residue is also essential for WaaL activity, which suggests a common mechanism for both enzymes, and supports the hypothesis that O-glycosylation and lipopolysaccharide (LPS) synthesis are evolutionarily related. These results contribute to the elucidation of the mechanism of O-OTases, which are promising targets for novel antibiotics and present an enormous potential for glycoengineering novel vaccines and therapeutics.

The innate reactivity of a membrane associated peptide towards lipids: acyl transfer to melittin without enzyme catalysis

The innate reactivity of the peptide melittin (H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH(2)) towards membrane lipids has been explored using LC-MS methods. The high sensitivity afforded by LC-MS analysis enabled acyl transfer to the peptide to be detected, within 4 h, from membranes composed of phosphocholines (PCs). Acyl transfer from PCs was also observed from mixtures of PC with phosphoserine (PS) or phosphoglycerol (PG). In the latter case, transfer from PG was also detected. The half-lives for melittin conversion varied between 24 h and 75 h, being fastest for POPC and slowest for DOPC/DMPG mixtures. The order of reactivity for amino groups on the peptide was N-terminus > K23 ≫ K21 > K7. Products arising from double-acylation of melittin were detected as minor components, together with a putative component derived from transesterification involving S18 of the peptide.

In Silico Model Escherichia Coli Membranes: Simulating a Lipid with a Cyclopropane Ring

Bacterial membranes are composed mostly of lipids with phosphoethanolamine (PE) and phosphoglycerol (PG) head groups and a variety of fatty acid chains. A defining characteristic of bacterial membranes is the presence of lipids with a cyclic-containing chain. To our knowledge, a phospholipid with such a chain has never been studied in a molecular dynamics simulation. Thus, novel force field parameters to describe a cyclopropane moiety were developed using parameters from the CHARMM36 (C36) general force field as a basis and high-level quantum mechanical energies. Two simple membranes and one complex membrane for use as realistic model Escherichia coli (E. coli) cytoplasmic membranes were designed for future work with secondary active transporters expressed in E. coli. Compositions of the membranes were based on several different experimental methods using an E. coli K12 strain grown on Luria broth. One simple membrane consisted of 85 % PE 18:1/16:0 (POPE) and 15 % PG 18:1/16:0(POPG), and the other of only PE cy17:0/16:0 (PMPE). The complex membrane consisted of six different phospholipids, the most prevalent being the cyclic-containing lipid, PMPE. NPT simulations were carried out at 310 K for 50 ns using the C36 lipid force field and the developed cyclopropane moiety force field for each membrane. NMR deuterium order parameters (SCD), density profiles, and diffusion constants are compared with experiment. The model membranes will provide a basis to study membrane bound proteins or other bound molecules expressed in E. coli with realistic molecular dynamic simulations.

Interactions with Host Cells Causes Neisseria meningitidis Pili to Become Unglued

Interactions with Host Cells Causes Neisseria meningitidis Pili to Become Unglued

Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Role of Different Phospholipids in the Synthesis of Pearl-Necklace-Type Gold−Silver Bimetallic Nanoparticles as Bioconjugate Materials

A seed-growth (S-G) method has been used to synthesize gold (Au) and Au−silver (Ag) bimetallic pear-necklace-type nanoparticles (NP) as bioconjugate materials by using a series of phospho-glycerol (PG) and phospho-choline (PC) lipids as capping agents. All PG lipids produce a fine pear-necklace arrangement of Au NP with dimensions between 10 and 20 nm. Addition of Ag converts this arrangement into Au−Ag bimetallic NP with a diameter essentially close to that of Au NP. Use of PC lipids does not show this arrangement but promotes a significant anisotropic growth especially in the presence of Ag. This difference has been attributed to a difference in the capping ability of PG and PC lipids because of their anionic and zwitterionic nature, respectively. XPS results have demonstrated the presence of adsorbed PG and PC lipids on Au or Au−Ag bimetallic surfaces in their respective samples. The results also indicate a decrease in the capping amount of a lipid with an increase in the growth of Au−Ag bimetallic NP....

Bolaamphiphiles promote phospholipid translocation across vesicle membranes.

A series of membrane-spanning bolaamphiphiles (molecules with two hydrophilic end groups connected by a hydrophobic linker) were prepared by a modular synthetic method and evaluated for their abilities to affect the dynamics of a surrounding bilayer membrane. The goal was to determine if the bolaamphiphiles promote the translocation of phospholipids across vesicle membranes. The bolaamphiphiles were incorporated at low levels (up to 5 mol %) in vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Inward translocation assays were performed using fluorescent, NBD-labeled phospholipid probes with phosphocholine (PC) or phosphoglycerol (PG) headgroups. The membrane-spanning bolaamphiphiles promote the translocation of both phospholipid probes in the order PG > PC, whereas shorter bolaamphiphiles (structures that must adopt a U-shape and keep both end groups in the same leaflet of the membrane), and regular amphiphiles with one hydrophilic end group, are inactive. These results are an exception to the rule-of-thumb that membrane-spanning bolaamphiphiles are inherently membrane-stabilizing molecules that inhibit all types of membrane transport.

Influence of Head Group Mismatch on the Miscibility of Phospholipids in the Physiologically-Relevant Fluid Phase: A Nearest-Neighbor Recognition Analysis

The mixing behavior of phosphoethanolamine (PE)-like and phosphoglycerol (PG)-like lipids have been examined in the fluid bilayer state by use of nearest-neighbor recognition methods [Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838]. Disulfide-based phospholipid dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) have been synthesized, containing PE- and PG-like head groups. Subsequent exchange and equilibration of the monomer units, via thiolate−disulfide interchange, produced a nonstatistical distribution of dimers in the fluid bilayer phase (60 °C); i.e., a heterodimer/homodimer ratio of 1.76 ± 0.05 was formed. Analogous membranes that were rich in cholesterol (i.e., 29 mol %) produced a statistical mixture of dimers. Equilibration experiments that were carried out using PE-like and PG-like lipids derived from 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and DMPE, respectively, gave similar results, except that a greater level of nearest-neigh...