Hydrophobic Acyl Chains Research Articles

A molecular mechanism for how pressure induces interdigitation of phospholipid bilayer membranes

The phase transition from the ripple gel phase to the interdigitated gel phase of bilayers of phosphatidylcholines (PCs) with two saturated long-chain fatty acids under high pressure was investigated by pressure-scanning microscopy, fluorometry, and dynamic light scattering (DLS) measurements. Microscopic observation for giant vesicles (GVs) of distearoyl-PC (DSPC) under high pressure showed that spherical GVs transforms significantly into warped and distorted spherical ones instantaneously at the pressure-induced interdigitation. The fluorescence intensities of amphiphilic probe Prodan and hydrophobic probe Laurdan in the dipalmitoyl-PC (DPPC) bilayer steeply decreased and increased, respectively, at the interdigitation, suggesting that the conformational change of the polar head group of DPPC molecule in the bilayer transiently occurred at the interdigitation. Further, it was found from the high-pressure DLS measurements that the size of the vesicle particles of the DPPC and DSPC transiently increases near the interdigitation pressure, whereas the chemically induced interdigitation by adding ethanol to the DSPC bilayer membrane under atmospheric pressure produce no such change in the particle size. Taking account of the critical packing parameter of the PC molecule, the above experimental results would lead us to the conclusion that the pressure-induced interdigitation is attributable to the increase in repulsive interaction between the polar head groups of the PC molecules resulting from the orientational change of the head group from a parallel alignment to a perpendicular one with respect to the bilayer surface by applying pressure, namely the transient state: it occurs when the repulsive interaction exceeds a threshold value for the balance between the repulsive interaction and the attractive interaction among the hydrophobic acyl chains.

Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?

Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts, together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and molecular dynamics simulations prove unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. The lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM-membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but calcium binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role-it not only forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer increasing the exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB2 FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within β-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.

Assessment of DPPC Liposome Disruption by Embedded Tocopheryl Malonate

In this study, the effect of α-tocopheryl malonate (TM) on physical and structural properties of DPPC liposomes was investigated using ANS fluorescence, DPH, and TMA–DPH anisotropy fluorescence and differential scanning calorimetry (DSC) methods. The presence of embedded TM in DPPC liposomes caused alteration in its phase transition temperatures, structural order, dynamics, and hydration of head groups increasingly with growing TM concentration. The ANS fluorescence results demonstrated that increasing TM presence in the DPPC gel phase due to interrupted membrane structure caused the formation of new binding sites. Temperature investigations in the range of 20 °C to 60 °C showed that increasing temperature rises ANS fluorescence which reaches local and global maxima at 36 °C and 42 °C, respectively. The rising TM concentration at the phase transition temperature of DPPC led to the lowering of ANS fluorescence, indicating a decreased binding of ANS. Simultaneously, during heating, a roughly 10-nm shift of ANS emission maximum was observed. The results indicated that in the fluid phase, the observed quenching appears as a result of increasing accessibility of water molecules into ANS in this region. The DPH results indicated that in the gel phase presence of TM introduced disorder in the hydrophobic acyl chain region led to its fluidization. The TMA–DPH results indicated an increasing disorder in the interface region and an increasing hydration of head group atoms at the surface of the membrane. The increasing concentration of TM results in the formation of multicomponent DSC traces, suggesting the formation of another structural phase. The applied methods proved that the incorporation of TM into DPPC membrane results in the interaction of malonate moiety with DPPC head group atoms in the interphase layer and induces the interruption in the membrane packing order, leading to its structural changes. The presented results show that TM presence could regulate the membrane properties, thus it may indicate one of the possible mechanisms responsible for the effective disruption of cell membranes by TM. The knowledge of molecular mechanism how TM interacts with the membrane will help to elucidate its possible pharmacological activity.

A rising tide lifts all MBOATs: recent progress in structural and functional understanding of membrane bound O-acyltransferases.

Acylation modifications play a central role in biological and physiological processes. Across a range of biomolecules from phospholipids to triglycerides to proteins, introduction of a hydrophobic acyl chain can dramatically alter the biological function and cellular localization of these substrates. Amongst the enzymes catalyzing these modifications, the membrane bound O-acyltransferase (MBOAT) family occupies an intriguing position as the combined substrate selectivities of the various family members span all three classes of these biomolecules. MBOAT-dependent substrates are linked to a wide range of health conditions including metabolic disease, cancer, and neurodegenerative disease. Like many integral membrane proteins, these enzymes have presented challenges to investigation due to their intractability to solubilization and purification. However, over the last several years new solubilization approaches coupled with computational modeling, crystallography, and cryoelectron microscopy have brought an explosion of structural information for multiple MBOAT family members. These studies enable comparison of MBOAT structure and function across members catalyzing modifications of all three substrate classes, revealing both conserved features amongst all MBOATs and distinct architectural features that correlate with different acylation substrates ranging from lipids to proteins. We discuss the methods that led to this renaissance of MBOAT structural investigations, our new understanding of MBOAT structure and implications for catalytic function, and the potential impact of these studies for development of new therapeutics targeting MBOAT-dependent physiological processes.

The Influence of Cationic Nitrosyl Iron Complex with Penicillamine Ligands on Model Membranes, Membrane-Bound Enzymes and Lipid Peroxidation.

This paper shows the biological effects of cationic binuclear tetranitrosyl iron complex with penicillamine ligands (TNIC-PA). Interaction with a model membrane was assessed using a fluorescent probes technique. Antioxidant activity was studied using a thiobarbituric acid reactive species assay (TBARS) and a chemiluminescence assay. The catalytic activity of monoamine oxidase (MAO) was determined by measuring liberation of ammonia. Antiglycation activity was determined fluometrically by thermal glycation of albumine by D-glucose. The higher values of Stern-Volmer constants (KSV) obtained for the pyrene located in hydrophobic regions (3.9 × 104 M-1) compared to KSV obtained for eosin Y located in the polar headgroup region (0.9 × 104 M-1) confirms that TNIC-PA molecules prefer to be located in the hydrophobic acyl chain region, close to the glycerol group of lipid molecules. TNIC-PA effectively inhibited the process of spontaneous lipid peroxidation, due to additive contributions from releasing NO and penicillamine ligand (IC50 = 21.4 µM) and quenched luminol chemiluminescence (IC50 = 3.6 μM). High activity of TNIC-PA in both tests allows us to assume a significant role of its radical-scavenging activity in the realization of antioxidant activity. It was shown that TNIC-PA (50-1000 μM) selectively inhibits the membrane-bound enzyme MAO-A, a major source of ROS in the heart. In addition, TNIC-PA is an effective inhibitor of non-enzymatic protein glycation. Thus, the evaluated biological effects of TNIC-PA open up the possibility of its practical application in chemotherapy for socially significant diseases, especially cardiovascular diseases.

Membrane Association Allosterically Regulates Phospholipase A2 Enzymes and Their Specificity.

Water-soluble proteins as well as membrane-bound proteins associate with membrane surfaces and bind specific lipid molecules in specific sites on the protein. Membrane surfaces include the traditional bilayer membranes of cells and subcellular organelles formed by phospholipids. Monolayer membranes include the outer monolayer phospholipid surface of intracellular lipid droplets of triglycerides and various lipoproteins including HDL, LDL, VLDL, and chylomicrons. These lipoproteins circulate in our blood and lymph systems and contain triglycerides, cholesterol, cholesterol esters, and proteins in their interior, and these are sometimes interspersed on their surfaces. Similar lipid-water interfaces also occur in mixed micelles of phospholipids and bile acids in our digestive system, which may also include internalized triglycerides and cholesterol esters. Diacyl phospholipids constitute the defining molecules of biological membranes. Phospholipase A1 (PLA1) hydrolyzes phospholipid acyl chains at the sn-1 position of membrane phospholipids, phospholipase A2 (PLA2) hydrolyzes acyl chains at the sn-2 position, phospholipase C (PLC) hydrolyzes the glycerol-phosphodiester bond, and phospholipase D (PLD) hydrolyzes the polar group-phosphodiester bond. Of the phospholipases, the PLA2s have been the most well studied at the mechanistic level. The PLA2 superfamily consists of 16 groups and numerous subgroups, and each is generally described as one of 6 types. The most well studied of the PLA2s include extensive genetic and mutational studies, complete lipidomics specificity characterization, and crystallographic structures. This Account will focus principally on results from deuterium exchange mass spectrometric (DXMS) studies of PLA2 interactions with membranes and extensive molecular dynamics (MD) simulations of their interactions with membranes and specific phospholipids bound in their catalytic and allosteric sites. These enzymes either are membrane-bound or are water-soluble and associate with membranes before extracting their phospholipid substrate molecule into their active site to carry out their enzymatic hydrolytic reaction. We present evidence that when a PLA2 associates with a membrane, the membrane association can result in a conformational change in the enzyme whereby the membrane association with an allosteric site on the enzyme stabilizes the enzyme in an active conformation on the membrane. We sometimes refer to this transition from a "closed" conformation in aqueous solution to an "open" conformation when associated with a membrane. The enzyme can then extract a single phospholipid substrate into its active site, and catalysis occurs. We have also employed DXMS and MD simulations to characterize how PLA2s interact with specific inhibitors that could lead to potential therapeutics. The PLA2s constitute a paradigm for how membranes interact allosterically with proteins, causing conformational changes and activation of the proteins to enable them to extract and bind a specific phospholipid from a membrane for catalysis, which is probably generalizable to intracellular and extracellular transport and phospholipid exchange processes as well as other specific biological functions. We will focus on the four main types of PLA2, namely, the secreted (sPLA2), cytosolic (cPLA2), calcium-independent (iPLA2), and lipoprotein-associated PLA2 (Lp-PLA2) also known as platelet-activating factor acetyl hydrolase (PAF-AH). Studies on a well-studied specific example of each of the four major types of the PLA2 superfamily demonstrate clearly that protein subsites can show precise specificity for one of the phospholipid hydrophobic acyl chains, often the one at the sn-2 position, including exquisite sensitivity to the number and position of double bonds.

Structural basis of substrate recognition and translocation by human very long-chain fatty acid transporter ABCD1.

Human ABC transporter ABCD1 transports very long-chain fatty acids from cytosol to peroxisome for β-oxidation, dysfunction of which usually causes the X-linked adrenoleukodystrophy (X-ALD). Here, we report three cryogenic electron microscopy structures of ABCD1: the apo-form, substrate- and ATP-bound forms. Distinct from what was seen in the previously reported ABC transporters, the two symmetric molecules of behenoyl coenzyme A (C22:0-CoA) cooperatively bind to the transmembrane domains (TMDs). For each C22:0-CoA, the hydrophilic 3’-phospho-ADP moiety of CoA portion inserts into one TMD, with the succeeding pantothenate and cysteamine moiety crossing the inter-domain cavity, whereas the hydrophobic fatty acyl chain extends to the opposite TMD. Structural analysis combined with biochemical assays illustrates snapshots of ABCD1-mediated substrate transport cycle. It advances our understanding on the selective oxidation of fatty acids and molecular pathology of X-ALD.

Fluidity is the way to life: lipid phase separation in bacterial membranes.

A hallmark of biological membranes is the dynamic localization of lipids and proteins. Lipids respond to temperature reduction below a critical point with phase separation, and poikilothermic animals and also bacteria adapt their lipid content to prevent gel phase formation in membranes. In a new study, Gohrbandt etal (2022) show that reduced membrane fluidity in bacterial cells causes reversible phase separation without membrane rupture invivo, highlighting the physical robustness of biological membranes.

HDL maturation and remodelling

Cholesterol in the circulation is mostly transported in an esterified form as a component of lipoproteins. The majority of these cholesteryl esters are produced in nascent, discoidal high density lipoproteins (HDLs) by the enzyme, lecithin:cholesterol acyltransferase (LCAT). Discoidal HDLs are discrete populations of particles that consist of a phospholipid bilayer, the hydrophobic acyl chains of which are shielded from the aqueous environment by apolipoproteins that also confer water solubility on the particles. The progressive LCAT-mediated accumulation of cholesteryl esters in discoidal HDLs generates the spherical HDLs that predominate in normal human plasma. Spherical HDLs contain a core of water insoluble, neutral lipids (cholesteryl esters and triglycerides) that is surrounded by a surface monolayer of phospholipids with which apolipoproteins associate. Although spherical HDLs all have the same basic structure, they are extremely diverse in size, composition, and function. This review is concerned with how the biogenesis of discoidal and spherical HDLs is regulated and the mechanistic basis of their size and compositional heterogeneity. Current understanding of the impact of this heterogeneity on the therapeutic potential of HDLs of varying size and composition is also addressed in the context of several disease states.

Interaction of a short antimicrobial peptide on charged lipid bilayer: A case study on aurein 1.2 peptide

Aurein 1.2 (aurein) is a short but active α-helical antimicrobial peptide discovered in Australian tree frogs (Litoria aurea). It shows inhibition on a broad spectrum of bacteria and cancer cells. With well-defined helicity, amphipathicity, and cationic charges, it readily binds to membranes and causes membrane change and disruption. This study provides details on how aurein interacts with charged lipid membranes by using neutron membrane diffraction (NMD) and neutron spin echo (NSE) spectroscopy on complex peptide-membrane systems. NMD provides higher resolution lipid bilayer structures than solution scattering. NMD revealed the peptide is mostly associated in the lipid headgroup region. Even at moderately high concentrations (e.g., peptide:lipid ratio of 1:30), aurein is located at the acyl chain-headgroup region without deep penetration into the hydrophobic acyl chain. However, it does reduce the elasticity of the membrane at that concentration, which was corroborated by the NSE results. Furthermore, NSE shows that aurein first softens the membrane, like many other α-helical peptides at low concentration, but then makes the membrane much more rigid, even without membrane pore formation. Combining our previous studies, the evidence shows that aurein at relatively low concentrations still modifies lipid distribution significantly and can cause membrane thinning and lateral segregation of charged lipids. At the same time, the membrane's mechanical properties are modified with much slower lipid diffusion. This suggests that aurein can attack the microbial membrane without the need to form membrane pores or disintegrate membranes; instead, it promotes the formation of domains at low concentration.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Effect of packing density of lipid vesicles on the Aβ42 fibril polymorphism

The aggregation of amyloid-β 1–42 (Aβ42) on lipid membranes is closely related to the pathology of Alzheimer’s disease (AD). Herein, we demonstrated the effect of the packing density of lipid vesicles on the Aβ42 fibrillation kinetics and fibril morphology. We used three distinct phosphatidylcholine (PC) lipids, containing different numbers of cis-double bonds in acyl chains, and therefore, a different packing density in the lipid vesicles. Our results showed that the fibrillation of Aβ42 was greatly enhanced and the formed fibrils became shorter as the number of double bonds in lipids increased. Due to the low-density characteristics of dioleoyl phosphatidylcholine (DOPC), Aβ42 monomers were able to interact with the hydrophobic acyl chain of lipids exposed to the aqueous phase, thereby inducing rapid fibrillation and short fibril morphologies. Furthermore, the effects of the anionic lipids dioleoyl phosphatidylserine (DOPS) and dioleoyl phosphatidylglycerol (DOPG), and mixed vesicles of DOPC/DOPS and DOPC/DOPG on Aβ42 fibrillations were investigated. The tight binding of Aβ42 to the lipid head groups via electrostatic interactions was able to suppress the modulation of Aβ42 fibrillations compared to accelerated fibrillations on loosely packed membranes. Our proposed mechanism regarding the influence of lipid packing density on Aβ42 fibrillations provides an advanced understanding of lipid-associated amyloid fibrillations.

Recent advances in analytical strategies for mass spectrometry-based lipidomics

Lipids are vital biological molecules and play multiple roles in cellular function of mammalian organisms such as cellular membrane anchoring, signal transduction, material trafficking and energy storage. Driven by the biological significance of lipids, lipidomics has become an emerging science in the field of omics. Lipidome in biological systems consists of hundreds of thousands of individual lipid molecules that possess complex structures, multiple categories, and diverse physicochemical properties assembled by different combinations of polar headgroups and hydrophobic fatty acyl chains. Such structural complexity poses a huge challenge for comprehensive lipidome analysis. Thanks to the great innovations in chromatographic separation techniques and the continuous advances in mass spectrometric detection tools, analytical strategies for lipidomics have been highly diversified so that the depth and breadth of lipidomics have been greatly enhanced. This review will present the current state of mass spectrometry-based analytical strategies including untargeted, targeted and pseudotargeted lipidomics. Recent typical applications of lipidomics in biomarker discovery, pathogenic mechanism and therapeutic strategy are summarized, and the challenges facing to the field of lipidomics are also discussed.

Octenidine: Novel insights into the detailed killing mechanism of Gram-negative bacteria at a cellular and molecular level

Octenidine (OCT) is a widely used antiseptic molecule with an antimicrobial spectrum covering a broad range of bacteria and fungi. However, the detailed molecular mechanism of killing has not yet been elucidated. The objective of our study was to investigate the mode of action of OCT's potent effect on Gram-negative bacteria using Escherichia coli as a model organism as well as corresponding model membranes. The effects of OCT on cellular morphology were observed by electron microscopy, changes affecting membrane integrity (surface charge, fluidity, permeabilisation and depolarisation) by zeta potential, fluorescence microscopy and spectroscopy. Specific interactions of OCT with membrane phospholipids were addressed using differential scanning calorimetry, X-ray scattering and fluorescence techniques. OCT neutralises the surface charge of E. coli leading to disruption of the outer membrane and dramatic loss of the cell wall and further penetrates through the periplasmic space reaching the inner membrane. Model membranes showed that OCT inserts into the hydrophobic fatty acyl chain region of the bilayer, inducing complete lipid disorder. The loss of membrane integrity is also reflected by membrane depolarisation and changes in membrane fluidity as shown by electron microscopy. Insertion of OCT into the outer and inner membrane of E. coli results in a chaotic lipid arrangement that leads to rapid disruption of the cell envelope. We propose that this unspecific mode of action based on purely physical interactions is the basis of the very broad antimicrobial profile and makes it unlikely that resistance to OCT will develop.

Interactions of anticancer drugs doxorubicin and idarubicin with lipid monolayers: New insight into the composition, structure and morphology

We quantify directly here for the first time the extents of interactions of two different anthracycline drugs with pure and mixed lipid monolayers with respect to the surface pressure and elucidate differences in the resulting interaction mechanisms. The work concerns interactions of doxorubicin (DOx) and idarubicin (IDA) with monolayers of the zwitterionic DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and negatively charged DMPS (1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt)) as well as a 7:3 mixture of the two lipids. These drugs are used in current cancer treatments, while the lipid systems were chosen as phosphocholines are the major lipid component of healthy cell membranes, and phosphoserines are the major lipid component that is externalized into the outer leaflet of cancerous cell membranes. It is shown that DOx interacts with DMPS monolayers to a greater extent than with DMPC monolayers by lower limits of a factor of 5 at a surface pressure of 10 mN/m and a factor of 12 at 30 mN/m. With increasing surface pressure, the small amount of drug (~0.3 µmol/m2) bound to DMPC monolayers is excluded from the interface, yet its interaction with DMPS monolayers is enhanced until there is even more drug (~3.2 µmol/m2) than lipid (~2.6 µmol/m2) at the interface. Direct evidence is presented for all systems studied that upon surface area compression lipid is reproducibly expelled from the monolayer, which we infer to be in the form of drug-lipid aggregates, yet the nature of adsorption of material back to the monolayer upon expansion is system-dependent. At 30 mN/m, most relevant to human physiology, the interactions of DOx and IDA are starkly different. For DOx, there is a conformational change in the interfacial layer driven by aggregation, resulting in the formation of lateral domains that have extended layers of drug. For the more lipophilic IDA, there is penetration of the drug into the hydrophobic acyl chain region of the monolayer and no indication of lateral segregation. In addition to the Langmuir technique, these advances were made as a result of direct measurements of the interfacial composition, structure and morphology using two different implementations of neutron reflectometry and Brewster angle microscopy. The results provide new insight into key processes that determine the uptake of drugs such as limited drug penetration through cell membranes by passive diffusion as well as activation of drug removal mechanisms related to multidrug resistance.

Hybrid-Supported Bilayers Formed with Mixed-Charge Surfactants on C18-Functionalized Silica Surfaces.

Mixtures of cationic-anionic surfactants have been shown to spontaneously form ordered monolayers at hydrophobic-hydrophilic boundaries, including air-water and oil-water interfaces. In this work, confocal Raman microscopy is used to investigate the structure of hybrid-supported surfactant bilayers (HSSBs) formed by deposition of a distal leaflet of mixed cationic-anionic surfactants onto a proximal leaflet of n-alkane (C18) chains on the interior surfaces of chromatographic silica particles. The surface coverage of the two surfactants in a hybrid bilayer was determined from carbon analysis and the relative Raman scattering of their respective head-groups. Within the measurement uncertainty, the stoichiometric ratio of the two surfactants is one-to-one, equivalent to mixed-charge-surfactant monolayers at air-water and oil-water interfaces and consistent with the role of the head-group electrostatic interactions in their formation. When self-assembled on the hydrophobic surface, pairs of oppositely charged n-alkyl chain surfactants resemble a phospholipid (phosphatidylcholine) molecule, with its zwitterionic head-group and two hydrophobic acyl chain tails. Indeed, the structure of these hybrid-supported surfactant bilayers on C18-modified silica surfaces is similar to that of hybrid-supported lipid bilayers (HSLBs) on the same supports, but with denser and more-ordered n-alkyl chains. Hybrid-supported surfactant bilayers exhibit a melting phase transition (gel to liquid-crystalline phase) with structural and energetic characteristics similar to those of hybrid-supported bilayers prepared from a zwitterionic phospholipid of the same alkyl chain length. These mixed-charge surfactants on n-alkane-modified silica are stable in water over time (months), results that suggest the potential use of these hybrid bilayers for generating supported lipid-bilayer-like surfaces or for separation applications.

Protocols for Enzymatic Fluorometric Assays to Quantify Phospholipid Classes.

Phospholipids, consisting of a hydrophilic head group and two hydrophobic acyl chains, are essential for the structures of cell membranes, plasma lipoproteins, biliary mixed micelles, pulmonary surfactants, and extracellular vesicles. Beyond their structural roles, phospholipids have important roles in numerous biological processes. Thus, abnormalities in the metabolism and transport of phospholipids are involved in many diseases, including dyslipidemia, atherosclerosis, cholestasis, drug-induced liver injury, neurological diseases, autoimmune diseases, respiratory diseases, myopathies, and cancers. To further clarify the physiological, pathological, and molecular mechanisms and to identify disease biomarkers, we have recently developed enzymatic fluorometric assays for quantifying all major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol + cardiolipin, and sphingomyelin. These assays are specific, sensitive, simple, and high-throughput, and will be applicable to cells, intracellular organelles, tissues, fluids, lipoproteins, and extracellular vesicles. In this review, we present the detailed protocols for the enzymatic fluorometric measurements of phospholipid classes in cultured cells.

Solid-state NMR structural investigations of peptide-based nanodiscs and of transmembrane helices in bicellar arrangements.

The membrane topology of the peptide 18A, a derivative of apolipoprotein A-I, is investigated in structural detail. Apolipoprotein A-I is the dominant protein component of high density lipoproteins with important functions in cholesterol metabolism. 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2) was designed to mimic the structure of tandem domains of class A amphipathic helices and has served as a lead peptide for biomedical applications. At low peptide-to-lipid ratios 18A partitions into phosphatidylcholine membranes with helix topologies parallel to the membrane surface, an alignment that is maintained when disc-like bicelles form at higher peptide-to-lipid ratios. Notably, the bicelles interact cooperatively with the magnetic field of the NMR spectrometer, thus the bilayer normal is oriented perpendicular to the magnetic field direction. A set of peptides that totals four 15N or 2H labelled positions of 18A allowed the accurate analysis of tilt and azimuthal angles relative to the membrane surface under different conditions. The topology agrees with a double belt arrangement forming a rim that covers the hydrophobic fatty acyl chains of the bicelles. In another set of experiments, it was shown that POPC nanodiscs prepared in the presence of diisobutylene/maleic acid (DIBMA) polymers can also be made to align in the magnetic field. Finally, the transmembrane domains of the DQ alpha-1 and DQ beta-1 subunits of the major histocomptability complex (MHC) class II have been prepared and reconstituted into magnetically oriented bicelles for NMR structural analysis.

Synthetic asymmetric lipid-like organosilanes for liposomal nanohybrid cerasomes toward potential medical applications

The design and synthesis of lipidic organoalkoxysilanes with different higher fatty acids as hydrophobic acyl chains for the construction of bilayer vesicles called ‘cerasomes’ were accomplished and their properties, including hydrodynamic diameter, zeta potential and encapsulation efficiency of water-soluble doxorubicin, were determined. Experiments have shown that an asymmetric hydrophobic block makes it possible to obtain cerasomes with acceptable size and stability. At the same time, the drug loading is quite high.