Lipid Conformation Research Articles

Using Molecular Dynamics Simulations to Elucidate a Role for Bacterial Ceramides

Sphingolipids synthesis was thought to be rare in Gram-negative bacteria, previously only found in a handful of taxa. We recently discovered ceramides in Caulobacter crescentus and demonstrated that these lipids play an important role in antibiotic and phage sensitivity. However, the mechanism by which ceramides affect resistance to antimicrobials, as well as their effects on the integrity of the cell membrane are not yet clear. In this study, a coarse-grained molecular dynamics simulation of a prototypical bacterial outer membrane is used to observe changes in the conformation of the outer membrane lipids in the presence of ceramides.

Determination of modifications in rat liver due to phthalate uptake by SAM, RS, and ICP-OES.

The use of phthalates as plasticizers has been omnipresent, especially in cosmetics and food packaging, despite the proven effects on some organs of humans and animals. Therefore, alterations in living organisms due to phthalate exposure attract the attention of many scientists. Here, we demonstrate a mechanical and chemical investigation of the mentioned effects of di(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP) on rat liver by utilizing scanning acoustic microscopy (SAM), Raman spectroscopy (RS) and inductively coupled plasma optical emission spectrometry (ICP-OES) for the first time in the literature, as far as we know. The combined analysis gives insights into the degree of modification in the tissue components and which chemicals lead to these modifications. Our study shows that the acoustic impedance values of tissues of DEHP and DBP delivered mother rats are higher than those of tissues of the control mother rat, while the acoustic impedance values of tissues of offspring rats of DEHP and DBP delivered mother rats do not differ significantly from those of tissues of the control offspring rats of the control mother rat. Besides, RS analysis shows how the incorporation of DEHP into liver tissues changes the configuration and conformation of lipids and fatty acids. ICP-OES results show increased element levels within the tissues of DEHP and DBP delivered rats. Therefore, we can say that phthalates cause modifications within the liver. This study is a preliminary effort to investigate tissues with a mechano-chemical probe.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Arrangement of Ceramides in the Skin: Sphingosine Chains Localize at a Single Position in Stratum Corneum Lipid Matrix Models.

Understanding the structure of the stratum corneum (SC) is essential to understand the skin barrier process. The long periodicity phase (LPP) is a unique trilayer lamellar structure located in the SC. Adjustments in the composition of the lipid matrix, as in many skin abnormalities, can have severe effects on the lipid organization and barrier function. Although the location of individual lipid subclasses has been identified, the lipid conformation at these locations remains uncertain. Contrast variation experiments via small-angle neutron diffraction were used to investigate the conformation of ceramide (CER) N-(tetracosanoyl)-sphingosine (NS) within both simplistic and porcine mimicking LPP models. To identify the lipid conformation of the twin chain CER NS, the chains were individually deuterated, and their scattering length profiles were calculated to identify their locations in the LPP unit cell. In the repeating trilayer unit of the LPP, the acyl chain of CER NS was located in the central and outer layers, while the sphingosine chain was located exclusively in the middle of the outer layers. Thus, for the CER NS with the acyl chain in the central layer, this demonstrates an extended conformation. Electron density distribution profiles identified that the lipid structure remains consistent regardless of the lipid’s lateral packing phase, this may be partially due to the anchoring of the extended CER NS. The presented results provide a more detailed insight on the internal arrangement of the LPP lipids and how they are expected to be arranged in healthy skin.

Selective and sensitive spectral signals on confocal Raman spectroscopy for detection of ex vivo skin lipid properties

AbstractSpectral signals of stratum corneum (SC) acquired from confocal Raman spectroscopy (CRS) are employed to track lipid variations and the most sensitive and efficient signals are of high interest. Herein, 1% of sodium lauryl sulfate treated SC was compared to water‐treated one to evaluate statistical difference of spectral signals concerning lipid content, SC thickness and lipid molecular structures. Results showed that peak ratio of 1425 to 1490 cm−1 and 1630 to 1710 cm−1 in fingerprint region and Gaussian‐function deconvoluted lipid‐keratin peak ratio in high wavenumber (HWN) region were both responsive for analyzing lipid content. SC thickness measurement using the full width of half‐maximum (FWHM) of 2920 to 2960 cm−1 signal presented good sensitivity. Nevertheless, the shift of peak 2850 cm−1 indicated less sensitivity compared to trans‐gauche peak signals and 1300 cm−1 peak position for lipid conformation analysis. Meanwhile, the ratio of peak 2880 and 2850 cm−1 indicating lipid lateral packing structure presented to be less responsive. More strikingly, FWHM of peak 2850 cm−1 appeared to be unable for lipid lateral packing order analysis in our study. In summary, this research can serve as a pilot study for further investigation of direct drug delivery and interaction between topical applied compounds and skin components.

A New Lipid Force Field (FUJI).

To explore inhomogeneous and anisotropic systems such as lipid bilayers, the Lennard-Jones particle mesh Ewald (LJ-PME) method has been applied without a conventional isotropic dispersion correction. As the popular AMBER and CHARMM lipid force fields were developed using a cutoff scheme, their lipid bilayers unacceptably shrink when using the LJ-PME method. In this study, a new all-atom lipid force field (FUJI) was developed on the basis of the AMBER force-field scheme including the Lipid14 van der Waals parameters. Point charges were calculated using the restrained electrostatic potentials of many lipid conformers. Further, torsion energy profiles were calculated using high-level ab initio molecular orbitals (LCCSD(T)/Aug-cc-pVTZ//LMP2/Aug-cc-pVTZ), following which the molecular mechanical dihedral parameters were derived through a fast Fourier transform. By incorporation of these parameters into a new lipid force field without fitting experimental data, the desired lipid characteristics such as the area per lipid and lateral diffusion coefficients were obtained through GROMACS molecular dynamics simulations using the LJ-PME method and virtual hydrogen sites. The calculated area per lipid and lateral diffusion coefficients showed satisfactory agreement with experimental data. Furthermore, the electron-density profiles along the membrane normal were calculated for pure lipid bilayers, and the resulting membrane thicknesses agreed well with the experimental values. As the new lipid force field is compatible with FUJI for protein and small molecules, the new FUJI force field will offer accurate modeling for complex systems consisting of various membrane proteins and lipids.

Interaction of chitosan derivatives with cell membrane models in a biologically relevant medium

HypothesisThe interaction of chitosan, a natural biopolymer with various biomedical applications, with lipid Langmuir films has been widely investigated as a simple model for cell membranes. However, to ensure polymer solubility, up to now only acidic subphases with pH significantly below biological fluids have been used. To increase the biological significance of these investigations, here we evaluated the effects of two chitosan derivatives (low molecular weight − CH, and positively charged − CH-P40) on phospholipid films (either zwitterionic DPPC or anionic DPPG) using phosphate buffered saline solutions (PBS) as a subphase. ExperimentsSurface pressure − area (π−A) isotherms were used to evaluate the expansion and changes in film elasticity, while Sum-Frequency Generation (SFG) vibrational spectroscopy provided information about the chain conformation of lipids. FindingsIt was found that chitosans caused a small expansion of the DPPC film by its insertion within the monolayer. In contrast, they distinctly expanded DPPG monolayers by both chitosan insertion within the lipid monolayer and by interacting with the anionic head group. Therefore, PBS buffer can be used as a subphase for more biologically relevant studies of chitosan interactions with Langmuir films, shedding light on why chitosan is antibacterial but not toxic to mammals, as the interaction mechanism depends on lipid headgroup charge.

Systematic Investigation of the Effect of Non-Ionic Emulsifiers on Skin by Confocal Raman Spectroscopy-A Comprehensive Lipid Analysis.

Non-ionic emulsifiers are commonly found in existing pharmaceutical and cosmetic formulations and have been widely employed to enhance the penetration and permeation of active ingredients into the skin. With the potential of disrupting skin barrier function and increasing fluidity of stratum corneum (SC) lipids, we herein examined the effects of two kinds of non-ionic emulsifiers on intercellular lipids of skin, using confocal Raman spectroscopy (CRS) with lipid signals on skin CRS spectrum. Non-ionic emulsifiers of polyethylene glycol alkyl ethers and sorbitan fatty acid esters were studied to obtain a deep understanding of the mechanism between non-ionic emulsifiers and SC lipids. Emulsifier solutions and dispersions were prepared and applied onto excised porcine skin. Water and sodium laureth sulfate solution (SLS) served as controls. SC lipid signals were analysed by CRS regarding lipid content, conformation and lateral packing order. Polyethylene glycol (PEG) sorbitan esters revealed no alteration of intercellular lipid properties while PEG-20 ethers appeared to have the most significant effects on reducing lipid content and interrupting lipid organization. In general, the polyoxyethylene chain and alkyl chain of PEG derivative emulsifiers might indicate their ability of interaction with SC components. HLB values remained critical for complete explanation of emulsifier effects on skin lipids. With this study, it is possible to characterize the molecular effects of non-ionic emulsifiers on skin lipids and further deepen the understanding of enhancing substance penetration with reduced skin barrier properties and increased lipid fluidity.

Investigating the interactions of corona-free SWCNTs and cell membrane models using sum-frequency generation.

The understanding of the interactions between biomolecules and nanomaterials is of great importance in many areas of nanomedicine and bioapplications. Numerous studies in this area have been performed. However, toxicological aspects involving the interaction between phospholipids and carbon nanotubes (CNTs) remain undefined, especially for those cases in which a protein corona is not formed around the nanomaterial (corona-free nanomaterials). This study focuses on the interaction of Langmuir films of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC) with corona-free, single-walled CNTs. Surface pressure-area isotherms and sum-frequency generation (SFG) vibrational spectroscopy, a non-linear optical technique used to study surfaces and interfaces, were used to investigate the lipid tail orientation and conformation, aiming to understand the interactions between phospholipids and single walled carbon nanotubes functionalized by carboxylic acid (SWCNTs-COOH) at the air-water interface under low ionic strength conditions. Data from isotherms and SFG spectra revealed that the SWCNT adsorption at the air-water interface is induced by the presence of both lipids, although at a lesser extent for DPPG due to its anionic head group, which could result in repulsion of SWCNTs-COOH that also bear a negative charge. Furthermore, lipid monolayers remained conformationally ordered, indicating insertion of SWCNTs into the lipid monolayer. Our results corroborate previous works and simulations in the literature, but made it possible to perform an in-depth investigation of the interaction of these nanomaterials with components of phospholipid membranes.

Differences in Meibum and Tear Lipid Composition and Conformation.

The compositional, structural, and functional relationships of meibum may provide insights into the loss of tear film stability. Although the conformation of meibum lipids has been studied rigorously, that of tear lipids has not. Tear lipids (TLHSCT) and meibum (MHSCT) from patients who had hematopoietic stem cell transplantation were pooled prospectively. The infrared spectra of meibum from donors with (MMGD) and without (Mn) meibomian gland dysfunction were retrospectively analyzed to measure the lipid composition and structure. The infrared CH stretching region was used to measure the relative content of CH3 and CH2 moieties in the meibum. The 3 major findings of the current study are as follows: 1) compared with Mn, MHSCT and MMGD had 18% fewer CH3 moieties; 2) compared with MHSCT, the phase transition temperature, cooperativity, and order were approximately 20% greater for TLHSCT; and 3) compared with Mn and MMGD, MHSCT and TLHSCT contained fewer double bonds. Tear lipids are more ordered than meibum lipids, which could have functional consequences. The human meibum peak height ratio of the CH3/CH2 bands is not a factor related to tear film stability with age or sex. The amount of CH3 moieties relative to CH2 moieties and saturation could contribute to a higher meibum lipid order associated with a younger age, meibomian gland dysfunction, and dry eye from hematopoietic stem cell transplantation. Therefore, the hydrocarbon order may be a marker of or contribute to an unstable tear film layer.

Structural Differences in Meibum From Donors After Hematopoietic Stem Cell Transplantations

Meibum is considered to be a key component of tears that serve to protect the eye, and conformational changes in meibum have not been studied extensively within the population of patients who had hematopoietic stem cell transplantation (HSCT). The aim of this study was to determine possible lipid conformational changes in the meibum of patients who had HSCT. Participants who had HSCT were randomly sampled for this prospective comparative study. Control participants did not have dry eye or had not undergone allogeneic or autologous stem cell transplantation. Fourier-transform infrared spectroscopy was used to measure meibum phase transition. Meibum was collected from both eyes of 36 donors without dry eye (Mc) and from 22 patients who had undergone HSCT (MHSCT). There were no significant differences between the phase transition parameters based on gender or race. The following were the significant differences (P < 0.0001) between the parameters for Mc compared with MHSCT : lipid order (% trans) at 33.4°C increased from 40 (1) to 54 (2), cooperativity decreased from 7.9 (0.4) to 5.4 (0.3), the phase transition temperature (C) increased from 30.3 (0.4) to 34.2 (0.9), and the magnitude of the phase transition (cm) increased from 4.0 (0.1) to 4.7 (0.5) (standard error of the mean). Conformational and thermodynamic differences were observed between Mc and MHSCT. The changes observed in the lipid conformation of meibum from patients receiving HSCTs suggest that meibum composition changes after stem cell transplantation, and clinicians should consider treating the meibomian glands to improve the ocular surface.

FTIR-based spectrum of salivary exosomes coupled with computational-aided discriminating analysis in the diagnosis of oral cancer.

To determine the Fourier-transform infrared (FTIR) spectra of salivary exosomes from oral cancer (OC) patients and healthy individuals (HI) and to assess its diagnostic potential using computational-aided models. Whole saliva samples were collected from 21 OC patients and 13 HI. Exosomes were pelleted using differential centrifugation (12,000g, 120,000g). The mid-infrared (IR) absorbance spectra (900-5000cm- 1 range) were measured using MIR8025 Oriel Fourier-transform IR equipped with a PIKE MIRacle ZnSe attenuated total reflectance attachment. Machine learning techniques, utilized to build discrimination models for the absorbance data of OC and HI, included the principal component analysis-linear discriminant analysis (PCA-LDA) and support vector machine (SVM) classification. Sensitivity, specificity and the area under the receiver operating characteristic curve were calculated. IR spectra of OC were consistently different from HI at 1072cm- 1 (nucleic acids), 2924cm- 1 and 2854cm- 1 (membranous lipids), and 1543cm- 1 (transmembrane proteins). The PCA-LDA discrimination model correctly classified the samples with a sensitivity of 100%, specificity of 89% and accuracy of 95%, and the SVM showed a training accuracy of 100% and a cross-validation accuracy of 89%. We showed the specific IR spectral signature for OC salivary exosomes, which was accurately differentiated from HI exosomes based on detecting subtle changes in the conformations of proteins, lipids and nucleic acids using optimized artificial neural networks with small data sets. This non-invasive method should be further investigated for diagnosis of oral cancer at its very early stages or in oral lesions with potential for malignant transformation.

Effect of ethanol adaption on the inactivation of Acetobacter sp. by pulsed electric fields

The effects of low concentrations of ethanol (3% to 9%, v/v) on cell membrane fatty acid composition, membrane lipid conformation, membrane fluidity and pulsed electric fields (PEF) resistance of Acetobacter sp. in the stationary phase were investigated. Results showed that the growth of Acetobacter sp. was inhibited, and analysis of gas chromatography–mass spectrometry, Raman spectroscopy and fluorescence polarization indicated that membrane fluidity of Acetobacter sp. cells was significantly increased by modifying membrane fatty acid composition and membrane lipid conformation after exposure to ethanol. Moreover, results showed that cells cultivated in the medium with relatively high ethanol concentrations were more vulnerable to PEF. For example, the inactivation of Acetobacter sp. (109 CFU/mL) cultivated with 9% ethanol by PEF (20.0 kV/cm, 6.0 ms) could reach 5.17 log, significantly higher than those cultivated without ethanol (3.22 log). This was confirmed by morphological analysis, showing various changes on the cell surface after PEF treatment. Acetobacter sp. is one of spoilage microorganisms in winemaking, and it is able to convert the ethanol produced by yeasts into acetic acid and thereby increase volatile acidity in wine. In this study, the effect of ethanol as a growth substrate on PEF resistance in Acetobacter sp. cells was investigated. Results here reported will contribute to better understand the influencing mechanisms of ethanol on the inactivation of Acetobacter sp. cells by PEF treatment and further develop the application of PEF for inactivating acetic acid bacteria (AAB) in the wine industry.

Quantification of Lipid Phase Order of In Vivo Human Skin Using Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy and Multivariate Curve Resolution Analysis.

A new analysis methodology utilizing multivariate curve resolution (MCR) has been successfully combined with Fourier transform infrared (FT-IR) measurement of in vivo human skin to resolve lipid phase constituents in the spectra relative to high and low chain ordering. A clinical study was performed to measure lipid order through different depths of stratum corneum of human subjects. Fourier transform IR spectra were collected through the top 10 layers of the skin on four sites on the left and right forearm of 12 individuals. Depth profiling was achieved by tape stripping to remove layers of skin with 10 successive tapes from each site. In vivo ATR FT-IR spectra were collected after removing each tape. Three isolated spectral regions were analyzed, centered around 2850 cm-1, 1460-1480 cm-1, and 730 cm-1, corresponding to stretching, scissoring, and rocking -CH2 vibrational modes, respectively. Both traditional lipid conformation analysis and MCR analysis were performed on the same spectral data. The lipid order ratio, expressed as the fraction of highly ordered orthorhombic (OR) lipids to the total lipids content (orthorhombic + hexagonal [HEX] + liquid crystal [LC]), was assessed as function of depth. Lipid order depth profiles (LODP) show an increase in order with the stratum corneum depth which can be adequately described by an exponential function for the data obtained in this study. The LODP derived from the three vibrational modes show very similar trends, although the absolute order ratios are somewhat different. The variance of the skin LODP across individuals is much greater than between sites within the same individual. The higher arm sites closer to the elbow on the left and right arm show no statistically significant difference and are recommended for use in comparative studies. The scissoring mode shows the highest sensitivity for determination of LODP value.

Lipid organization in xerosis: the key of the problem?

Although xerosis is a common skin disorder among the population, there is no invivo global study focusing on xerotic skin. Hence, the objective of this study was to characterize xerotic skin from the surface to the molecular scale with invivo and non-invasive approaches. For this purpose, 15 healthy volunteers with normal skin and 19 healthy volunteers with xerotic skin were selected by a dermatologist, thanks to a visual scorage. Firstly, the skin surface was characterized with biometric measurements. Then, the state of skin dryness was assessed by invivo confocal microscopy. The molecular signature of xerotic skin was then determined by invivo confocal Raman microspectroscopy. Finally, an identification of stratum corneum (SC) lipids was performed using Normal phase liquid chromatography (NP-LC) coupled to two detectors: Corona and High Resolution/Mass Spectroscopy (HR/MS). Results obtained at the skin surface displayed an increase in the transepidermal water loss (TEWL) and a decrease in the hydration rate in xerotic skin. Confocal microscopy revealed an alteration of the cell shape in xerotic skin. Moreover, confocal Raman microspectroscopy demonstrated directly invivo and non-invasively the lack of organization and conformation of lipids in this skin. Finally, HPLC analyses revealed that the three ceramide sub-classes (NdS, NS and EOP) significantly decrease in xerosis. Altogether, these results identify parameters for the characterization of xerotic skin compared to normal. This study highlighted discriminative parameters from the surface to the molecular level invivo and non-invasively between xerotic and normal skins. These results will be useful for the development of new cosmetic active ingredients dedicated to xerotic skin.

Insights into Tear Film Stability from Babies and Young Adults: A Study of Human Meibum Lipid Conformation and Rheology.

Babies have the most stable tears and people with dry eye have the least stable tears. Meibum may contribute to tear film stability, so in this study, the hydrocarbon chain conformation and rheology of meibum from babies was studied for the first time. Infrared spectroscopy was used to measure lipid phase transitions. Rheology was measured using Langmuir film technology. Meibum from 25 donors 1 to 13 years old was compared with meibum from 18 donors 13 to 25 years old. The phase transition temperature and lipid order (stiffness) increased with increasing age from 1 to 25 years. The increase in meibum lipid order from 1 to 25 years of age may contribute to the instability of the tear film with age and contribute to films with a higher reciprocal compressibility modulus that are not as compressible and not as viscoelastic. Changes in the lipid phase transition parameters of meibum lipid with dry eye are an exacerbation of the changes observed with age. The lower reciprocal compressibility moduli of meibum films from children and babies compared with meibum from adults reiterates higher stability in their films which spread better, resist deformation, and facilitates their ability to be quickly restored after blinking.

Locating Carbon-Carbon Double Bonds in Unsaturated Phospholipids by Epoxidation Reaction and Tandem Mass Spectrometry.

The presence of carbon-carbon double bonds (C═Cs) in unsaturated phospholipids is closely related to lipid conformations and physiochemical activities. Previously, we have demonstrated that epoxidation reaction facilitated by low-temperature plasma (LTP) enabled the structural analysis of unsaturated fatty acids (FAs). Epoxidation of the C═C leads to the production of an epoxide, which can be easily cleaved via collision-induced dissociation (CID) to produce diagnostic ions indicative of the C═C bond locations in FAs. In this work, we further developed this method for analysis of phospholipids. Tandem mass spectrometry analysis with epoxidation reaction was performed in both positive and negative ion mode to analyze phosphatidylcholines (PCs), phosphatidic acids (PAs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The developed method was applied in a shotgun lipidomics approach to characterize phospholipids in a bovine liver extract.

Confocal Raman microspectroscopy as an alternative to differential scanning calorimetry to detect the impact of emulsifiers and formulations on stratum corneum lipid conformation

The purpose of this study was to investigate the impact of emulsifiers and formulations on stratum corneum (SC) lipid conformation and evaluate confocal Raman microspectroscopy (CRM) as an alternative method to differential scanning calorimetry (DSC) in this research context. To this end, four different formulations were used: three conventional creams that contained ionic and/or non-ionic emulsifiers and one surfactants-free emulsion stabilized by a polymeric emulsifier. Additionally, all emulsifiers were tested in aqueous solutions/dispersions in the respective concentrations as present in the formulations. In this study, emulsifiers and formulations were applied onto excised porcine skin during incubation in Franz diffusion cells. Subsequently, SC was isolated, dried and subjected to CRM and DSC measurement to analyse lipid structural changes after treatment. In CRM measurement, 1080 cm−1/(1130 cm−1 + 1070 cm−1) peak ratio, which represents the C-C skeleton vibration and trans-gauche conformation order of lipids, was investigated. Various emulsifiers and formulations showed different impact on SC lipid conformation. Specifically, cetearyl alcohol and sodium cetearyl sulfate mixture dispersion showed the strongest ability among all studied emulsifiers, followed by glycerol monostearate, polyoxyethylene-20-glycerol monostearate as well as their mixture. Polysorbate 60, cetyl stearyl alcohol and their mixture did not affect SC lipid structure. The results of CRM and DSC correlated very well, indicating CRM, as an alternative to DSC, can be a reliable method to investigate SC lipid conformation.

Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.

A New Method of Assessing Lipid Mixtures by 31P Magic-Angle Spinning NMR

A variety of lipids that differ by their chains and headgroups are found in biomembranes. In addition to studying the overall membrane phase, determination of the structure, dynamics, and headgroup conformation of individual lipids in the mixture would be of great interest. We have thus developed, to our knowledge, a new approach using solid-state 31P NMR, magic-angle spinning, and chemical-shift anisotropy (CSA) recoupling, using an altered version of the recoupling of chemical shift anisotropy (ROCSA) pulse sequence, here penned PROCSA. The resulting two-dimensional spectra allowed the simultaneous measurement of the isotropic chemical shift and CSA of each lipid headgroup, thus providing a valuable measure of its dynamics and structure. PROCSA was applied to mixtures of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in various relative proportions, to mimic bacterial membranes and assess the respective roles of lipids in shaping these bilayers. The results were interpreted in terms of membrane topology, lipid propensity to adopt various phases or conformations, and lipid-lipid miscibility. Our results showed that PG dictates the lipid behavior when present in a proportion of 20 mol % or more. A small proportion of PG is thus able to impose a bilayer structure to the hexagonal phase forming PE. We discuss the requirement for lipids, such as PE, to be able to adopt non-bilayer phases in a membrane.