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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Confocal Raman Microscopy for In-situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this paper, we employ in-situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayers deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically-trapped phospholipid vesicle membranes. Finally and additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2013

Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation

The human nuclear receptor liver receptor homolog-1 (LRH-1) has an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of diabetes and hepatic diseases. LRH-1 is known to bind phospholipids, but the role of phospholipids in controlling LRH-1 activation remains highly debated. Here we describe the structure of both apo LRH-1 and LRH-1 in complex with the antidiabetic phospholipid dilauroylphosphatidylcholine (DLPC). Together with hydrogen-deuterium exchange MS and functional data, our studies show that DLPC binding is a dynamic process that alters co-regulator selectivity. We show that the lipid-free receptor undergoes previously unrecognized structural fluctuations, allowing it to interact with widely expressed co-repressors. These observations enhance our understanding of LRH-1 regulation and highlight its importance as a new therapeutic target for controlling diabetes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Raman Microscopy Investigation of GLP-1 Peptide Association with Supported Phospholipid Bilayers

A wide range of important biological processes occur at phospholipid membranes including cell signaling, where a peptide or small molecule targets a membrane-localized receptor protein. In this work, we report the adaptation of confocal-Raman microscopy to quantify populations of unlabeled glucagon-like peptide-1 (GLP-1), a membrane-active 30-residue incretin peptide, in supported phospholipid bilayers deposited on the interior surfaces of wide-pore porous silica particles. Quantification of lipid bilayer-associated peptide is achieved by measuring the Raman scattering intensity of the peptide relative to that of the supported-lipid bilayer, which serves as an internal standard. The dependence of the bilayer-associated GLP-1 population on the solution-concentration of GLP-1 produces an isotherm used to determine the equilibrium constant for peptide-bilayer association and the maximum peptide surface coverage. The maximum coverage of GLP-1 in the lipid bilayer was found to be only 1/5th of a full monolayer based on its hydrodynamic radius. The saturation coverage, therefore, is not limited by the size of GLP-1 but by the ability of the bilayer to accommodate the peptide at high concentrations within the bilayer. Raman spectra show that GLP-1 association with the supported bilayer is accompanied by structural changes consistent with the intercalation of the peptide into bilayer, where the observed increase in acyl-chain order would increase the lipid density and provide free volume needed to accommodate the peptide. Furthermore, these results were compared with previous measurements of the association of fluorescently-labeled GLP-1 with a planar-supported bilayer; the unlabeled peptide exhibits a 3-fold greater affinity for the lipid-bilayer on the porous-silica support suggesting that the fluorescent label alters GLP-1 lipid-bilayer association.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell-membranes to model association reactions of proteins with lipid-bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal-Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers, deposited on the interior surfaces of wide-pore silica surfaces. We examine the question whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin-coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ~60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. Here, the results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.

36 MATERIALS SCIENCE↗
DOE OSTI2018

Confocal-Raman Microscopy Characterization of Supported Phospholipid Bilayers Deposited on the Interior Surfaces of Chromatographic Silica

A typical approach to exploring the structure and dynamics of biological membranes is through the deposition of model lipid bilayers on planar supports by Langmuir-trough or vesicle-fusion methods. Planar-supported lipid bilayers have been shown to exhibit structure and properties similar to those of lipid-vesicle membranes and are suitable for biosensing applications. Investigations using these planar-membrane models are limited to high-sensitivity methods capable of detecting a small population of molecules at the interface between a planar support and aqueous solution. In this work, we present evidence that supported-lipid bilayers can be deposited by vesicle fusion onto the interior surfaces throughout the wide-pore network of chromatographic silica particles. The thickness of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) film and headgroup spacing are consistent with a single bilayer of DMPC deposited onto the pore surfaces. The high specific surface area of these materials generates phospholipid concentrations easily detected by confocal-Raman microscopy within an individual particle, which allows the structure of these supported bilayers to be investigated. Raman spectra of porous-silica-supported DMPC bilayers are equivalent to spectra of DMPC vesicle membranes, both above and below their melting phase transitions, suggesting comparable phospholipid organization and bilayer structure. These porous-silica-supported model membranes could share benefits that planar-supported lipid bilayers bring to biosensing applications, but in a material that overcomes the limited surface area of a planar support. To try this concept, the potential of these porous-silica-supported lipid bilayers as high-surface-area platforms for label-free Raman-scattering-based protein biosensing is demonstrated with detection of concanavalin A selectively binding to a lipid-immobilized mannose target.

36 MATERIALS SCIENCE↗
DOE OSTI2019

Reply to “Comment on ‘Dynamics of Phospholipid Membranes beyond Thermal Undulation’”

Recently, Granek postulated a connection of the Brochard-Lennon (BL) “red blood cell” mode with the short time relaxation of liposomes observed by neutron spin echo (NSE) spectroscopy. The experimental observations themselves clearly exclude this BL mode as an explanation. Here, we emphasize that it is an inherent behavior of lipids detectable with different techniques.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2018

Dynamics of Phospholipid Membranes beyond Thermal Undulations

Here, we investigated the molecular dynamics of unilamellar liposomes by neutron spin echo spectroscopy. We report the first experimental evidence of a short-range motion at the length scale of the size of the headgroup of a lipid. The associated mean squared displacement shows a t 0.26 dependence in the pico- to nanosecond region that indicates another process beyond the predictions of the Zilman–Granek (ZG) model (t 0.66 ) and translational diffusion (t 1 ). A comparison with theory shows that the observed low exponent is associated with a non-Gaussian transient trapping of lipid molecules in a local area and supports the continuous time random walk model. The analysis of the mean squared displacement leads to the important conclusion that the friction at the interface between water and liposomes plays a minor role. Center of mass diffusion of liposomes and transient trapping of lipids define the range in which the ZG model can be applied to analyze membrane fluctuations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Saturated fatty acid in the phospholipid monolayer contributes to the formation of large lipid droplets

The degree of saturation of fatty acid chains in the bilayer membrane structure is known to control membrane fluidity and packing density. However, the significance of fatty acid composition in the monolayers of lipid droplets (LDs) has not been elucidated. In this study, we noted a relationship between the size of LDs and the fatty acid composition of the monolayer. To obtain large LDs, we generated NIH3T3 cells overexpressing fat-specific protein 27 (FSP27). This induced the fusion of LDs, resulting in larger LDs in FSP27-overexpressing cells compared with LDs in control cells. Moreover, the lipid extracts of LDs from FSP27-overexpressing cells reconstituted large-droplet emulsions in vitro, implying that the lipid properties of LDs might affect the size of LDs. FSP27-overexpressing cells had more saturated fatty acids in the phospholipid monolayer of the LDs compared with control cells. To further investigate the effects of the degree of phospholipid unsaturation on the size of LDs, we synthesized artificial emulsions of a lipid mixed with distearoylphosphatidylcholine (DSPC, diC18:0-PC) and with dioleoylphosphatidylcholine (DOPC, diC18:1n-9-PC) and compared the sizes of the resulting LDs. The emulsions prepared from saturated PC had larger droplets than those prepared from unsaturated PC. Our results suggest that saturated fatty acid chains in phospholipid monolayers might establish the form and/or stability of large LDs. - Highlights: • The lipid extracts of larger LDs from FSP27 cells reconstructed large-droplet emulsions. • Isolated LDs from FSP27 cells had more saturated fatty acids in the phospholipid monolayer compared with the control. • Saturated fatty acids in the phospholipid monolayer are a factor in the formation of large emulsions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

An engineered opsin monomer scrambles phospholipids

The G protein-coupled receptor opsin is a phospholipid scramblase that facilitates rapid transbilayer phospholipid exchange in liposomes. The mechanism by which opsin scrambles lipids is unknown. It has been proposed that lipid translocation may occur at protein-protein interfaces of opsin dimers. To test this possibility, we rationally engineered QUAD opsin by tryptophan substitution of four lipid-facing residues in transmembrane helix 4 (TM4) that is known to be important for dimerization. Atomistic molecular dynamics simulations of wild type and QUAD opsins combined with continuum modeling revealed that the tryptophan substitutions lower the energetically unfavorable residual hydrophobic mismatch between TM4 and the membrane, reducing the drive of QUAD opsin to dimerize. We purified thermostable wild type and QUAD opsins, with or without a SNAP tag for fluorescence labeling. Single molecule fluorescence measurements of purified SNAP-tagged constructs revealed that both proteins are monomers. Fluorescence-based activity assays indicated that QUAD opsin is a fully functional scramblase. However, unlike wild type opsin which dimerizes en route to insertion into phospholipid vesicles, QUAD opsin reconstitutes as a monomer. We conclude that an engineered opsin monomer can scramble phospholipids, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Interactions of amelogenin with phospholipids

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. In this paper, we investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin–cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (~334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder–order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Finally, our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Synthesis and Purification of Homogeneous Lipid-Based Peptide Nanocarriers by Overcoming Phospholipid Ester Hydrolysis

Despite the therapeutic promise of phospholipid-based nanocarriers, a major obstacle to their widespread clinical translation is a susceptibility to fatty acid ester hydrolysis, leading to lack of quality control and inconsistencies in self-assembly formulations. Using electrospray ionization mass spectrometry fragmentation in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have demonstrated a method to detect hydrolysis of one or both of the fatty acid esters in a PEGylated phospholipid, DSPE-PEG, in conditions commonly applied during nanocarrier production. Because such carriers are increasingly being used to deliver peptide-based therapeutics, we further investigated the hydrolysis of phospholipid esters in conditions used for solid-phase peptide synthesis and high-performance liquid chromatography of peptides. We ultimately detail a synthetic strategy to reliably produce pure phospholipid–peptide bioconjugates (peptide amphiphiles), while avoiding unintended or unnoticed hydrolyzed byproducts that could lead to polymorphic nanotherapeutics with dampened therapeutic efficacy. We believe that such an approach could help standardize phospholipid–peptide-based therapeutic development, testing, and clinical translation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Phospholipid Membrane Protection by Sugar Molecules during Dehydration-Insights into Molecular Mechanisms Using Scattering Techniques

Scattering techniques have played a key role in our understanding of the structure and function of phospholipid membranes. These techniques have been applied widely to study how different molecules (e.g., cholesterol) can affect phospholipid membrane structure. However, there has been much less attention paid to the effects of molecules that remain in the aqueous phase. One important example is the role played by small solutes, particularly sugars, in protecting phospholipid membranes during drying or slow freezing. In this paper, we present new results and a general methodology, which illustrate how contrast variation small angle neutron scattering (SANS) and synchrotron-based X-ray scattering (small angle (SAXS) and wide angle (WAXS)) can be used to quantitatively understand the interactions between solutes and phospholipids. Specifically, we show the assignment of lipid phases with synchrotron SAXS and explain how SANS reveals the exclusion of sugars from the aqueous region in the particular example of hexagonal II phases formed by phospholipids.

Garvey, Christopher J.↗
DOE OSTI2024

Quantifying Phospholipids in Organic Samples Using a Hydrophilic Interaction Liquid Chromatography–Inductively Coupled Plasma High-Resolution Mass Spectrometry (HILIC-ICP-HRMS) Method

Here, in this study, a novel method using hydrophilic interaction liquid chromatography (HILIC) coupled with inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) was introduced for the quantification of phospholipids in oil samples. The method employed a bridged ethyl hybrid (BEH) stationary phase HILIC column with a tetrahydrofuran (THF)/water mobile phase, enhancing the solubility and detection of phospholipids. During the study, a gradient/matrix effect on ICP-HRMS sensitivity was observed and successfully compensated for experimentally, ensuring reliable quantification results. This approach has proven effective for a wide range of different oil samples including vegetable oils, animal fats, and phospholipid supplements. Notably, this method allowed the direct quantification of phospholipids in oil samples, bypassing the need for prior sample preparation methods, such as solid phase extraction (SPE), thereby streamlining the analytical process. The precision, accuracy, and reduced need for extensive sample preparation offered by this method mark a significant advancement in lipids analysis. Its robustness and broad applicability have substantial implications for industries such as food and renewable energy production, where both efficient and accurate lipid identification and quantification are crucial.

09 BIOMASS FUELS↗
DOE OSTI2014

Multi-component modeling of quasielastic neutron scattering from phospholipid membranes

We investigated molecular motions in the 0.3–350 ps time range of D{sub 2}O-hydrated bilayers of 1-palmitoyl-oleoyl-sn-glycero-phosphocholine and 1,2-dimyristoyl-sn-glycero-phosphocholine in the liquid phase by quasielastic neutron scattering. Model analysis of sets of spectra covering scale lengths from 4.8 to 30 Å revealed the presence of three types of motion taking place on well-separated time scales: (i) slow diffusion of the whole phospholipid molecules in a confined cylindrical region; (ii) conformational motion of the phospholipid chains; and (iii) fast uniaxial rotation of the hydrogen atoms around their carbon atoms. Based on theoretical models for the hydrogen dynamics in phospholipids, the spatial extent of these motions was analysed in detail and the results were compared with existing literature data. The complex dynamics of protons was described in terms of elemental dynamical processes involving different parts of the phospholipid chain on whose motions the hydrogen atoms ride.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

General model of phospholipid bilayers in fluid phase within the single chain mean field theory

Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2013

Differential Intrahepatic Phospholipid Zonation in Simple Steatosis and Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) occurs frequently in a setting of obesity, dyslipidemia and insulin resistance, but the etiology of the disease, particularly the events favoring progression to nonalcoholic steatohepatitis (NASH) as opposed to simple steatosis (SS), are not fully understood. Based on known zonation patterns in protein, glucose and lipid metabolism, coupled with evidence that phosphatidylcholine may play a role in NASH pathogenesis, we hypothesized that phospholipid zonation exists in liver and that specific phospholipid abundance and distribution may be associated with histologic disease. A survey of normal hepatic protein expression profiles in the Human Protein Atlas revealed pronounced zonation of enzymes involved in lipid utilization and storage, particularly those facilitating phosphatidylcholine (PC) metabolism. Immunohistochemistry of obese normal, SS and NASH liver specimens with anti-phosphatidylethanomine Nmethyltransferase (PEMT) antibodies showed a progressive decrease in the zonal distribution of this PC biosynthetic enzyme. Phospholipid quantitation by liquid chromatography mass spectrometry (LC-MS) in hepatic extracts of Class III obese patients with increasing NAFLD severity revealed that most PC species with 32, 34 and 36 carbons as well as total PC abundance was decreased with SS and NASH. Matrix assisted laser desorption ionization - imaging mass spectrometry (MALDI-IMS) imaging revealed strong zonal distributions for 32, 34 and 36 carbon PCs in controls (minimal histologic findings) and SS that was lost in NASH specimens. Specific lipid species such as PC 34:1 and PC 36:2 best illustrated this phenomenon. These findings suggest that phospholipid zonation may be associated with the presence of an intrahepatic proinflammatory phenotype and thus have broad implications in the etiopathogenesis of NASH.

59 BASIC BIOLOGICAL SCIENCES↗