Acyl Chain Order Research Articles

Chirality-Dependent Interaction of d- and l-Menthol with Biomembrane Models.

Chirality plays a vital role in biological membranes and has a significant effect depending on the type and arrangement of the isomer. Menthol has two typical chiral forms, d- and l-, which exhibit different behaviours. l-Menthol is known for its physiological effect on sensitivity (i.e., a cooling effect), whereas d-menthol causes skin irritation. Menthol molecules may affect not only the thermoreceptors on biomembranes, but also the membrane itself. Membrane heterogeneity (lipid rafts, phase separation) depends on lipid packing and acyl chain ordering. Our interest is to elaborate the chirality dependence of d- and l-menthol on membrane heterogeneity. We revealed physical differences between the two optical isomers of menthol on membrane heterogeneity by studying model membranes using nuclear magnetic resonance and microscopic observation.

Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer.

Membrane proteins usually need to be extracted from their native environment and separated from other membrane components for in-depth in vitro characterization. The use of styrene/maleic acid (SMA) copolymers to solubilize membrane proteins and their surrounding lipids into bilayer nanodiscs is an attractive approach toward this goal. We have recently shown that a diisobutylene/maleic acid (DIBMA) copolymer similarly solubilizes model and cellular membranes but, unlike SMA(3:1), has a mild impact on lipid acyl-chain order and thermotropic phase behavior. Here, we used fluorescence spectroscopy, small-angle X-ray scattering, size-exclusion chromatography, dynamic light scattering, and 31P nuclear magnetic resonance spectroscopy to examine the self-association of DIBMA and its membrane-solubilization properties against lipids differing in acyl-chain length and saturation. Although DIBMA is less hydrophobic than commonly used SMA(3:1) and SMA(2:1) copolymers, it efficiently formed lipid-bilayer nanodiscs that decreased in size with increasing polymer/lipid ratio while maintaining the overall thickness of the membrane. DIBMA fractions of different molar masses were similarly efficient in solubilizing a saturated lipid. Coulomb screening at elevated ionic strength or reduced charge density on the polymer at low pH enhanced the solubilization efficiency of DIBMA. The free-energy penalty for transferring phospholipids from vesicular bilayers into nanodiscs became more unfavorable with increasing acyl-chain length and unsaturation. Altogether, these findings provide a rational framework for using DIBMA in membrane-protein research by shedding light on the effects of polymer and lipid properties as well as experimental conditions on membrane solubilization.

On the Calculation of Acyl Chain Order Parameters from Lipid Simulations.

For molecular dynamics simulations of biological membrane systems to live up to the potential of providing accurate atomic level detail into membrane properties and functions, it is essential that the force fields used to model such systems are as accurate as possible. One membrane property that is often used to assess force field accuracy is the carbon-hydrogen (or carbon-deuterium) order parameters of the lipid tails, which can be accurately measured using experimental NMR techniques. There are a variety of analysis tools available to calculate these order parameters from simulations and it is essential that these computational tools work correctly to ensure the accurate assessment of the simulation force fields. In this work we compare many of these computational tools for calculating the order parameters of POPC membranes. While tools that work on all-atom systems and tools that work on saturated lipid tails in general work extremely well, we demonstrate that the majority of the tested tools that calculate the order parameters for unsaturated united-atom lipid tails do so incorrectly. We identify tools that do perform accurate calculations and include one such program with this work, enabling rapid and accurate calculation of united-atom lipid order parameters. Furthermore, we discuss cases in which it is nontrivial to appropriately predict the unsaturated carbon order parameters in united-atom systems. Finally, we examine order parameter splitting for carbon 2 in sn-2 lipid chains, demonstrating substantial deviations from experimental values in several all-atom and united-atom lipid force fields.

In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

We report the application of NMR dynamic spectral editing for probing the structure and dynamics of molecular constituents in fresh, intact cells and in freshly prepared thylakoid membranes of Chlamydomonas reinhardtii (Cr.) green algae. For isotope labeling, wild-type Cr. cells were grown on 13C acetate-enriched minimal medium. 1D 13C J-coupling based and dipolar-based MAS NMR spectra were applied to distinguish 13C resonances of different molecular components. 1D spectra were recorded over a physiological temperature range, and whole-cell spectra were compared to those taken from thylakoid membranes, evaluating their composition and dynamics. A theoretical model for NMR polarization transfer was used to simulate the relative intensities of direct, J-coupling, and dipolar-based polarization from which the degree of lipid segmental order and rotational dynamics of the lipid acyl chains were estimated. We observe that thylakoid lipid signals dominate the lipid spectral profile of whole algae cells, demonstrating that with our novel method, thylakoid membrane characteristics can be detected with atomistic precision inside intact photosynthetic cells. The experimental procedure is rapid and applicable to fresh cell cultures, and could be used as an original approach for detecting chemical profiles, and molecular structure and dynamics of photosynthetic membranes in vivo in functional states.

Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy.

A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with the membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. A force spectroscopy experiment shows that PrP106-126 reduces Young's modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid atomic vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intrachain conformation, while the interchain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the interchain interaction, while the intrachain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs.

Bilayer Interactions among Unsaturated Phospholipids, Sterols, and Ceramide

Using differential scanning calorimetry and lifetime analysis of trans-parinaric acid fluorescence, we have examined how cholesterol and cholesteryl phosphocholine (CholPC) affect gel-phase properties of palmitoyl ceramide (PCer) in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC) bilayers. By 2H NMR, we also measured fluid-phase interactions among these lipids using deuterated analogs of POPC, PCer, and cholesterol. The PCer-rich gel phase in POPC bilayers (9:1 molar ratio of POPC to PCer) was partially and similarly dissolved (and thermostability decreased) by both cholesterol and CholPC (sterol was present equimolar to PCer, or in fourfold excess). In DOPC bilayers (4:1 DOPC/PCer molar ratio), CholPC was much more efficient in dissolving the PCer-rich gel phase when compared to cholesterol. This can be interpreted as indicating that PCer interaction with POPC was stronger than PCer interaction with DOPC. PCer-CholPC interactions were also more favored in DOPC bilayers compared to POPC bilayers. In the fluid POPC-rich phase, cholesterol increased the order of the acyl chain of d2-PCer much more than did CholPC. In DOPC-rich fluid bilayers, both cholesterol and CholPC increased d2-PCer acyl chain order, and the ordering induced by CholPC was more efficient in DOPC than in POPC bilayers. In fluid POPC bilayers, the ordering of 3-d1-cholesterol by PCer was weak. In summary, we found that in the gel phase, sterol effects on the PCer-rich gel phase were markedly influenced by the acyl chain composition of the fluid PC. The same was true for fluid-phase interactions involving the sterols. Our results further suggest that PCer did not display high affinity toward either of the sterols used. We conclude that the nature of unsaturated phospholipids (POPC versus DOPC) in bilayers has major effects on the properties of ceramide gel phases and on sterol-ceramide-phospholipid interactions in such complex bilayers.

Effects of seaweed sterols fucosterol and desmosterol on lipid membranes

Higher sterols are universally present in large amounts (20–30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.

Localization and Ordering of Lipids Around Aquaporin-0: Protein and Lipid Mobility Effects.

Hydrophobic matching, lipid sorting, and protein oligomerization are key principles by which lipids and proteins organize in biological membranes. The Aquaporin-0 channel (AQP0), solved by electron crystallography (EC) at cryogenic temperatures, is one of the few protein-lipid complexes of which the structure is available in atomic detail. EC and room-temperature molecular dynamics (MD) of dimyristoylglycerophosphocholine (DMPC) annular lipids around AQP0 show similarities, however, crystal-packing and temperature might affect the protein surface or the lipids distribution. To understand the role of temperature, lipid phase, and protein mobility in the localization and ordering of AQP0-lipids, we used MD simulations of an AQP0-DMPC bilayer system. Simulations were performed at physiological and at DMPC gel-phase temperatures. To decouple the protein and lipid mobility effects, we induced gel-phase in the lipids or restrained the protein. We monitored the lipid ordering effects around the protein. Reducing the system temperature or inducing lipid gel-phase had a marginal effect on the annular lipid localization. However, restraining the protein mobility increased the annular lipid localization around the whole AQP0 surface, resembling EC. The distribution of the inter-phosphate and hydrophobic thicknesses showed that stretching of the DMPC annular layer around AQP0 surface is the mechanism that compensates the hydrophobic mismatch in this system. The distribution of the local area-per-lipid and the acyl-chain order parameters showed particular fluid- and gel-like areas that involved several lipid layers. These areas were in contact with the surfaces of higher and lower protein mobility, respectively. We conclude that the AQP0 surfaces induce specific fluid- and gel-phase prone areas. The presence of these areas might guide the AQP0 lipid sorting interactions with other membrane components, and is compatible with the squared array oligomerization of AQP0 tetramers separated by a layer of annular lipids.

Link between Fluorescent Probe Partitioning and Molecular Order of Liquid Ordered-Liquid Disordered Membranes.

Fluorescence microscopy is an important technique for studying lipid membranes and is increasingly being used for examining liquid ordered-liquid disordered phase coexistence. Liquid-liquid phase coexistence is a phenomenon of biological interest because it led to the lipid raft hypothesis, which postulates the existence of lateral heterogeneities in cell membranes. Observation of membrane heterogeneity relies on differential distribution of fluorescent membrane markers, but this can also modify the phase behavior, complicating the observation. We have used 2H NMR to measure the physical changes to 35:35:30 (mol/mol) DOPC/DPPC-D62/chol membranes introduced by fluorescent probes Laurdan and naphthopyrene. We measured miscibility transition temperature (Tmix) and DPPC-D62 chain order for a range of probe concentrations. We found that up to 0.5 mol% of the equipartitioning probe Laurdan does not influence DPPC-D62 acyl chain order or phase behavior. In contrast, 2.0 mol% Laurdan slightly increases the fraction of DPPC-D62 in the liquid disordered phase below the Tmix and increases Tmix by 1 °C. Conversely, the nominally liquid ordered phase preferring probe naphthopyrene slightly perturbs the membrane even at concentrations as low as 0.3 mol%. This suggests that the strength of fluorescent probe partitioning between liquid ordered and liquid disordered phases correlates with the degree of perturbation to membrane phase behavior.

Membrane Insertion of a Dinuclear Polypyridylruthenium(II) Complex Revealed by Solid-State NMR and Molecular Dynamics Simulation: Implications for Selective Antibacterial Activity.

Dinuclear polypyridylruthenium(II) complexes bridged by a flexible methylene linker have received considerable interest as potential antibacterial agents. Their potency and uptake into bacterial cells is directly modulated by the length of the bridging linker, which has implicated membrane interactions as an essential feature of their mechanism of action. In this work, a combination of molecular dynamics (MD) simulations and solid-state NMR was used to present an atomistic model of a polypyridylruthenium(II) complex bound and incorporated into a bacterial membrane model. The results of 31P, 2H, 1H, and 13C NMR studies revealed that the antibacterial [{Ru(phen)2}2(μ-bb12)]4+ complex (Rubb12), where phen = 1,10-phenanthroline and bb12 = bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane), incorporated into a negatively charged model bacterial membrane, but only associated with the surface of a charge-neutral model of a eukaryotic membrane. Furthermore, an inactive [{Ir(phen)2}2(μ-bb12)]6+ (Irbb12) analogue, which is not taken up by bacterial cells, maintained only a surface-bound association with both bacterial and eukaryotic model membranes according to 31P and 2H NMR. The effects of Rubb12 on 31P chemical shift anisotropy and 2H acyl chain order parameters for negatively charged membranes correlated with a membrane-spanning state of the complex according to MD simulation-in which the metal centers embed in the lipid head group region and the central void, created by the biconic shape of the complex, resulting in increasing disorder of lipid acyl chains and membrane-thinning. A transbilayer mechanism and membrane-spanning may be essential for the cellular uptake and antibacterial activity of this class of compounds.

The rational design of biomimetic skin barrier lipid formulations using biophysical methods.

The focus of this communication was to study phospholipid-structured emulsions whose phase behaviour is modified with monoalkyl fatty amphiphiles. Ideally, these systems would mimic key physical and structural attributes observed in human stratum corneum (SC) so that they better alleviate xerotic skin conditions. Phosphatidylcholine-structured emulsions were prepared, and their phase behaviour modified with monoalkyl fatty amphiphiles. The effect of molecular volume, acyl chain length and head-group interactions was studied using a combination of physical methods. Water vapour transmission rate (WVTR) was used as a primary test to assess occlusive character. Changes in the vibrational modes observed in Fourier transform infrared (FTIR) spectroscopy and bilayer spacing measured by X-ray diffraction (XRD) were then applied to elucidate the lateral and lamellar microstructural characteristics in the systems. Water vapour transmission rate demonstrated that as the phosphatidylcholine acyl chain length increased from C14, to C18, to C22, there was a corresponding increase in occlusive character. The addition of monoalkyl fatty amphiphiles such as behenic acid, behenyl alcohol or cetostearyl alcohol to a base formulation incorporating dipalmitoyl and distearoylphosphatidylcholine (C18) was seen to further increase barrier characteristics of the emulsions. FTIR methods used to probe lipid-chain conformational ordering demonstrated that as phosphatidylcholine acyl chain lengths increased, there was a corresponding improvement in acyl chain ordering, with an increase in thermal transition temperatures. The addition of a monoalkyl fatty amphiphile resulted in conformational order and thermal transition temperature improvements trending towards those observed in stratum corneum. FTIR also demonstrated that systems containing behenic acid or behenyl alcohol exhibited features associated with orthorhombic character. X-ray diffraction data showed that addition of monoalkyl fatty amphiphile also resulted in thicker lamellar structures than when those agents are not present. The generalized approach described herein is shown to mechanistically describe the occlusive character of phospholipid-structured formulations in the presence of long-chain fatty acids or alcohols and that they exhibit characteristics mimicking those found in human SC lipids.

Changes in the Properties and Organization of Human Lens Lipid Membranes Occurring with Age

ABSTRACTPurpose: This research was undertaken to document the changes in the organization and properties of human lens lipid membranes that occur with age.Methods: Human lens lipid membranes prepared from the total lipids extracted from clear lens cortices and nuclei of donors from age groups 0–20 and 21–40 years were investigated. An electron paramagnetic resonance technique and nitroxide spin labels (analogues of phospholipids and cholesterol) were used.Results: Two distinct lipid domains, the phospholipid–cholesterol domain (PCD) and the pure cholesterol bilayer domain (CBD), were detected in all investigated membranes. Profiles of the acyl chain order, fluidity, hydrophobicity, and oxygen transport parameter across discriminated coexisting lipid domains were assessed. Independent of the age-related changes in phospholipid composition, the physical properties of the PCD remained the same for all age groups and were practically identical for cortical and nuclear membranes. However, the properties of pure CBDs changed significantly with the age of the donor and were related to the size of the CBD, which increased with the age of the donor and was greater in nuclear than in cortical membranes. A more detailed analysis revealed that the size of the CBD was determined mainly by the cholesterol content in the membrane.Conclusions: This paper presents data from four age groups: 0–20, 21–40, 41–60, and 61–70 years. Data from age groups 41–60 and 61–70 years were published previously. Combining the previously published data with those data obtained in the present work allowed us to show the changes in the organization of cortical and nuclear lens lipid membranes as functions of age and cholesterol. It seems that the balance between age-related changes in membrane phospholipid composition and cholesterol content plays an integral role in the regulation of cholesterol-dependent processes in fiber cell membranes and in the maintenance of fiber cell membrane homeostasis.

Hexagonal phase with ordered acyl chains formed by a short chain asymmetric ceramide

Ceramides constitute a group of lipids with usually high melting temperature that also favor negative curvature in membranes when mixed with other lipids. The short chain C10:0 ceramide is an asymmetric lipid which consists of an 18 carbon sphingosine base N-acylated with decanoic acid. According to high sensitivity differential scanning calorimetry, it shows a minor exothermic peak at 61°C and a main endothermic transition at 75°C. By small angle X-ray scattering and polarized light microscopy we found that, at temperatures below the main transition, the fully hydrated lipid dispersions are arranged in a tridimensional structure corresponding to an inverted hexagonal phase. Infrared spectroscopy and wide angle X-ray diffraction indicated that the acyl chains of ceramides exhibit a relatively high order in the hexagonal phase. As far as we know, this is the first report of a lipid hexagonal phase having highly ordered acyl chains. Molecular asymmetry due to the different length of the sphingosine and the N-acyl chains of C10:0 ceramide may explain why this novel phase is formed.

Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2H solid-state NMR

Nuclear magnetic resonance (NMR) is commonly used to probe the effect of antimicrobial agents on bacterial membranes using model membrane systems. Ideally, considering the complexity of membranes, the interaction of molecules with membranes should be studied in vivo. The interactions of two antimicrobial peptides (AMPs) with intact Escherichia coli and Bacillus subtilis were investigated using deuterium solid-state NMR. Specifically, we studied caerin 1.1 and aurein 1.2 isolated from the skin of Australian tree frogs. The minimal inhibitory concentration value for E. coli and B. subtilis was about 100μg/mL and 30μg/mL, respectively, for both peptides. A protocol to deuterate the membrane phospholipids of non-mutated B. subtilis was established using deuterated palmitic acid. 2H NMR spectra combined with spectral moment analysis support the interaction of the two AMPs with the hydrophobic core of the bacterial membranes. The presence of peptides decreased the order of the lipid acyl chains for both E. coli and B. subtilis, but at higher peptide concentrations for the Gram(+) bacteria. This may be explained by the presence of other cell wall components, such as the negatively-charged teichoic and lipoteichoic acids in the peptidoglycan, which would interact with the AMPs and decrease their actual concentration on the membrane surface. The mechanism of action of the AMPs thus depends on their local concentration as well as the membrane environment. The differences between the AMPs interaction with E. coli and B. subtilis reveal the importance of studying intact bacteria.

Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C18-Functionalized Chromatographic Particles

Measuring lipid-membrane partitioning of small molecules is critical to predicting bioavailability and investigating molecule-membrane interactions. A stable model membrane for such studies has been developed through assembly of a phospholipid monolayer on n-alkane-modified surfaces. These hybrid bilayers have recently been generated within n-alkyl-chain (C18)-modified porous silica and used in chromatographic retention studies of small molecules. Despite their successful application, determining the structure of hybrid bilayers within chromatographic silica is challenging because they reside at buried interfaces within the porous structure. In this work, we employ confocal Raman microscopy to investigate the formation and temperature-dependent structure of hybrid-phospholipid bilayers in C18-modified, porous-silica chromatographic particles. Porous silica provides sufficient surface area within a confocal probe volume centered in an individual particle to readily measure, with Raman microscopy, the formation of an ordered hybrid bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the surface C18 chains. The DMPC surface density was quantified from the relative Raman scattering intensities of C18 and phospholipid acyl chains and found to be ∼40% of a DMPC vesicle membrane. By monitoring Raman spectra acquired versus temperature, the bilayer main phase transition was observed to be broadened and shifted to higher temperature compared to a DMPC vesicle, in agreement with differential scanning calorimetry (DSC) results. Raman scattering of deuterated phospholipid was resolved from protonated C18 chain scattering, showing that the lipid acyl and C18 chains melt simultaneously in a single phase transition. The surface density of lipid in the hybrid bilayer, the ordering of both C18 and lipid acyl chains upon bilayer formation, and decoupling of C18 methylene C-H vibrations by deuterated lipid acyl chains all suggest an interdigitated acyl chain structure. The simultaneous melting of both layers is also consistent with an interdigitated structure, where immobility of surface-grafted C18 chains decreases the cooperativity and increases the melting temperature compared to a vesicle bilayer.

The Adherence Scale in Chronic Diseases (ASCD). The power of knowledge: the key to successful patient — health care provider cooperation

Introduction. Autotransfusions of ozonised blood or infusions of gaseous ozone into blood vessels andbody cavities are believed to exert therapeutic effects in some pathological states. Investigations on thereaction of ozone with biological molecules and membrane structures are a subject of crucial importance. The present study aimed to yield more precise data about the alterations, which occur in different erythrocytemembrane regions subjected to medical ozone. This could provide some additional informationabout the structural changes in the membrane at a molecular level. Material and methods. Blood was obtained from 22 healthy volunteers (aged 21 to 63) by vein puncture andmixed with 1/10 volume of 0.13 M trisodium citrate. Erythrocytes were isolated from fresh blood by centrifugationat 4°C, at 1,500 × g and purified by three cycles of resuspension and washing with PBS. Ozone was generatedby passing pure gaseous oxygen at 30 l/h through an apparatus producing silent electric discharges. EPRspectra were obtained at X-band (9.4 GHz), at modulation frequency of 100 kHz. The scan time was 4 min, and the time constant 0.3 s. Since biological membranes are heterogeneous systems composed of severalcoexisting domains, EPR spectra are superimpositions of several spectra with different fluidity parameters. Results. The effects of ozone at two concentrations (10 and 45 g/m3) on fluidity and phospholipid domainstructure of erythrocyte membranes were investigated by electron paramagnetic resonance (EPR). Atincreased ozone concentration (45 g/m3), the portion of the least ordered domains (WLO) increased, witha corresponding decrease of ordered (WMO, WO), more rigid regions. The order of lipid acyl chains of twoordered (MO and O) as well as the least ordered (LO) domains diminished, as expressed by smaller orderparameters. For ozone concentration of 10 g/m3 values of order parameter were slightly increased, whichindicates a tendency to rigidisation of lipid bilayer at this ozone concentration. This change was, however,statistically insignificant. There were no statistically significant differences in the thermotropic behaviour ofweight factor of ordered domains (WMO, WO) between ozonised and control red cells. Conclusion. The obtained data shows that ozonation of erythrocytes results in cell membrane fluidisationand an increase in red cell deformability. On the other hand, these results could suggest that ozonationof erythrocytes leads to structural changes in the membranes, especially in cytoskeletal proteins, but thiseffect is probably dose-dependent.

The two sides of a lipid-protein story.

Protein-membrane interactions play essential roles in a variety of cell functions such as signaling, membrane trafficking, and transport. Membrane-recruited cytosolic proteins that interact transiently and interfacially with lipid bilayers perform several of those functions. Experimental techniques capable of probing changes on the structural dynamics of this weak association are surprisingly limited. Among such techniques, electron spin resonance (ESR) has the enormous advantage of providing valuable local information from both membrane and protein perspectives by using intrinsic paramagnetic probes in metalloproteins or by attaching nitroxide spin labels to proteins and lipids. In this review, we discuss the power of ESR to unravel relevant structural and functional details of lipid-peripheral membrane protein interactions with special emphasis on local changes of specific regions of the protein and/or the lipids. First, we show how ESR can be used to investigate the direct interaction between a protein and a particular lipid, illustrating the case of lipid binding into a hydrophobic pocket of chlorocatechol 1,2-dioxygenase, a non-heme iron enzyme responsible for catabolism of aromatic compounds that are industrially released in the environment. In the second case, we show the effects of GPI-anchored tissue-nonspecific alkaline phosphatase, a protein that plays a crucial role in skeletal mineralization, and on the ordering and dynamics of lipid acyl chains. Then, switching to the protein perspective, we analyze the interaction with model membranes of the brain fatty acid binding protein, the major actor in the reversible binding and transport of hydrophobic ligands such as long-chain, saturated, or unsaturated fatty acids. Finally, we conclude by discussing how both lipid and protein views can be associated to address a common question regarding the molecular mechanism by which dihydroorotate dehydrogenase, an essential enzyme for the de novo synthesis of pyrimidine nucleotides, and how it fishes out membrane-embedded quinones to perform its function.

Lipid Interactions and Organization in Complex Bilayer Membranes

Bilayer lipids influence the lateral structure of the membranes, but the relationship between lipid properties and the lateral structure formed is not always understood. Model membrane studies on bilayers containing cholesterol and various phospholipids (PLs) suggest that high and low temperature melting PLs may segregate, especially in the presence of cholesterol. The effect of different PL headgroups on lateral structure of bilayers is also not clear. Here, we have examined the formation of lateral heterogeneity in increasingly complex (up to five-component) multilamellar bilayers. We have used time-resolved fluorescence spectroscopy with domain-selective fluorescent probes (PL-conjugated trans-parinaric acid), and 2H NMR spectroscopy with site or perdeuterated PLs. We have measured changes in bilayer order using such domain-selective probes both as a function of temperature and composition. Our results from time-resolved fluorescence and 2H NMR showed that in ternary bilayers, acyl chain order and thermostability in sphingomyelin-rich domains were not affected to any greater extent by the headgroup structure of the monounsaturated PLs (phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine) in the bilayer. In the complex five-component bilayers, we could not detect major differences between the different monounsaturated PLs regarding cholesterol-induced ordering. However, cholesterol clearly influenced deuterated N-palmitoyl sphingomyelin differently than the other deuterated PLs, suggesting that cholesterol favored N-palmitoyl sphingomyelin over the other PLs. Taken together, both the fluorescence spectroscopy and 2H NMR data suggest that the complex five-component membranes displayed lateral heterogeneity, at least in the lower temperature regimen examined.

Cholesterol Promotes the Peripheral Binding of Retroviral Proteins to Lipid Bilayers

Rous sarcoma virus (RSV) serves as a model retrovirus to study interactions of the structural protein, Gag, of enveloped viruses such as HIV, with the plasma membrane (PM). Gag-PM interactions are a critical step in the viral lifecycle and understanding their mechanisms is important for facilitating the development of retroviral therapies. Both in vitro and in vivo experiments have provided important insights into Gag's binding behavior but a much needed molecular picture of the protein-bilayer interactions is missing. It is known that the protein binds peripherally to the membrane via its 104 amino-acids-long membrane-binding domain (MBD). This interaction is primarily driven by electrostatics but multiple experiments have reported an additional enhancement of binding in the presence of cholesterol. This effect has been observed in both the myristoylated MBD of HIV, and the non-myristoylated MBD of RSV. To learn the mechanism of this effect we have examined the interactions of RSV MBD with cholesterol-rich and cholesterol-depleted bilayers composed of the charged and neutral lipids POPC and POPS with all-atom MD simulations. The results are consistent with the reported experimental findings, showing that the structure of the protein remains mostly intact, but its position with respect to the membrane changes depending on the lipid composition. Thus, residue-based analysis of protein-lipid interactions identifies up to 20% increase in Lys-POPS contacts in the cholesterol containing bilayer, whose area per lipid is more than 25% lower than that of the POPC/POPS bilayer without cholesterol. We have analyzed the effect of MBD on the lipids interacting directly with it in the two systems, and report on potential cholesterol-mediated bilayer properties (including lipid headgroup tilt, acyl chain order and surface charge density) that contribute to the enhanced binding.

Vitamin E Differentially Destabilizes Bilayer Structure of Docosahexaenoic and Oleic-Acid Containing Model Membranes

Vitamin E (α-tocopherol) is a lipid-soluble antioxidant that has a primary role of protecting polyunsaturated phospholipids from oxidation in membranes. Whether there is preferential interaction between α-tocopherol and polyunsaturated phospholipids to optimize this function has been a longstanding question. Here we compare the effect of α-tocopherol on the molecular organization of polyunsaturated 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (16:0-22:6PE, PDPE) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (16:0-18:1PE, POPE) in mixtures with sphingomyelin (SM). By solid-state 2H NMR spectroscopy, we directly observed POPE-d31 and PDPE-d31 (analogs of POPE and PDPE with a perdeuterated sn-1 chain) in the mixed membranes. In supplementary X-ray diffraction experiments, we further probed membrane architecture. At higher temperatures (>20 °C), the spectra observed for POPE-d31 in POPE/SM/α-tocopherol (2:2:1 mol) and for PDPE-d31 in PDPE/SM/α-tocopherol (2:2:1 mol) consist of a superposition of two spectral components that we ascribe to α-tocopherol promoting a transition from lamellar (Lα) to inverted hexagonal (HII) phase. This interpretation is confirmed by X-ray diffraction data. Analysis of depaked spectra shows that the amount of HII phase increases with temperature. The increase ranges from 7 to 28% between 31 and 55 °C for POPE-d31, while for PDPE-d31 the increase is less and ranges from 6 to 20% over the same temperature range. A more modest increase in acyl chain order is also seen for PDPE-d31 (3%) than POPE-d31 (8%) remaining in the Lα phase (37 °C). The molecular origin of the differential in bilayer stability between the two mixed membranes will be discussed in terms of the greater disorder of PDPE and its ability to accommodate α-tocopherol.