Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy

Lipid droplets (LDs), present in many cell types, are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs). With the discovery of new LD functions (e.g., in immune response, protein clearage, and occurrence with disease), new methods to study LD chemical composition in situ are necessary. We present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy that allows deciphering LD TAG composition in different biochemically complex samples with submicrometer spatial resolution. Employing a set of standard TAGs, we generate a spectral training matrix capturing the variation caused in Raman-like spectra by TAG backbone, chain length, and number of double bonds per chain, as well as the presence of proteins or other diluting molecules. Comparing our fitting approach to gas chromatography measurements for mixtures of standard TAGs and food oils, we find the root mean-square error for the prediction of TAG chemistry to be 0.69 CH₂ and 0.15 #C=C. When progressing to more complex samples such as oil emulsions and LDs in various eukaryotic cells, we find good agreement with bulk gas chromatography measurements. For differentiated adipocytes, we find a significant increase in the number of double bonds in small LDs (below 2 μm in diameter) compared to large LDs (above 2 μm in diameter). Coupled with a relatively limited sample preparation requirement, this approach should enable rapid and accurate TAG LD analysis for a variety of cell biology and technological applications.     

Partitioning of Tetrachlorophenol into Lipid Bilayers and Sarcoplasmic Reticulum: Effect of Length of Acyl Chains,Carbonyl Group of Lipids and Biomembrane Structure

We report results of a partitioning study of 2,3,4,6-tetrachlorophenol (TeCP). In the study we explored (1) the effect of the length of acyl chains of lipids (C16:1 – C24:1) and alkanes (C6–C16), (2) the role of the carbonyl group of lipids, and (3) the effect of molecular structure of the sarcoplasmic reticulum membrane on TeCP partitioning. Mole fraction partition coefficients have been measured using equilibrium dialysis for un-ionized (HA), and ionized (A) species, Kp_x^HA, Kp_x^A. Their values are concentration-dependent. Partition coefficients were analyzed in terms of a model that accounts for saturation of membrane associated with the finite area of partition site, and electrostatic interactions of (A-) species with charged membrane. Limiting values of partition coefficients, corresponding to infinite dilution of solute, Kp_x0^HA, Kp_x0^A were obtained. Kp_x0^HA and Kp_x0^A measure the strength of solute-membrane interactions. Studies were done with single-layered vesicles of lipids with variable chain length: 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (C16:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1), 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22:1), and 1 ,2-dinervonoyl-sn-glycero-3-phosphocholine (C24:1), and egg-PC. Kp_x0 for transfer of TeCP from water into lipid membranes was found to be independent of the length of acyl chains, whereas Kp_x0 for transfer from water into alkanes increased with the length of alkane. The effect of the carbonyl CO group of lipids on partitioning was measured using 1,2-di-o-octadecenyl-sn-glycero-3-phosphocholine (CO absent) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (CO present) liposomes. Carbonyl groups, known to change dipolar potential, had no effect on partitioning. Partition coefficients of un-ionized and ionized forms of TeCP were invariant to the presence of proteins and other membrane components of sarcoplasmic reticulum (SR) membrane.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

Telomeric Overhang Length Determines Structural Dynamics and Accessibility to Telomerase and ALT-Associated Proteins

1. Download : Download high-res image (194KB)
2. Download : Download full-size image

     

Effects of Thimerosal on Lipid Bilayers and Human Erythrocytes: An In Vitro Study

Thimerosal (THI, ethyl-mercury thiosalicylate) is added to vaccines as a preservative; as a consequence, infants may have been exposed to bolus doses of Hg that collectively added up to nominally 200 µg Hg during the first 6 months of life. While several studies report an association between THI-containing vaccines and neurological disorders, other studies do not support the causal relation between THI and autism. With the purpose to understand the molecular mechanisms of the toxic effect of THI it was assayed on human red cells and in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids found in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of THI to interact with DMPC and DMPE was determined by X-ray diffraction and differential scanning calorimetry, whereas intact human erythrocytes were observed by optical, defocusing and scanning electron microscopy. The experimental findings of this study demonstrated that THI interacted in a concentration-dependent manner with DMPC and DMPE bilayers, and in vitro interacted with erythrocytes inducing morphological changes. However, concentrations were considerable higher than those present in vaccines.     

Orientation and Order of the Amide Group of Sphingomyelin in Bilayers Determined by Solid-State NMR

Sphingomyelin (SM) and cholesterol (Chol) are considered essential for the formation of lipid rafts; however, the types of molecular interactions involved in this process, such as intermolecular hydrogen bonding, are not well understood. Since, unlike other phospholipids, SM is characterized by the presence of an amide group, it is essential to determine the orientation of the amide and its order in the lipid bilayers to understand the nature of the hydrogen bonds in lipid rafts. For this study, 1′-¹³C-2-¹⁵N-labeled and 2′-¹³C-2-¹⁵N-labeled SMs were prepared, and the rotational-axis direction and order parameters of the SM amide in bilayers were determined based on ¹³C and ¹⁵N chemical-shift anisotropies and intramolecular ¹³C-¹⁵N dipole coupling constants. Results revealed that the amide orientation was minimally affected by Chol, whereas the order was enhanced significantly in its presence. Thus, Chol likely promotes the formation of an intermolecular hydrogen-bond network involving the SM amide without significantly changing its orientation, providing a higher order to the SM amide. To our knowledge, this study offers new insight into the significance of the SM amide orientation with regard to molecular recognition in lipid rafts, and therefore provides a deeper understanding of the mechanism of their formation.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.     

Effects of Chain Length and Saturability of Fatty Acids on Phospholipids and Proteins in Plasma Membranes of Bovine Mammary Gland

Free fatty acids (FFAs) in plasma are essential substrates for de novo synthesis of milk fat, or directly import into mammary cells. The physico-chemical properties of mammary cells membrane composition affected by FFAs with different chain lengths and saturability are unclear yet. Employing GC, FTIR and fluorescence spectroscopy, the adsorption capacity, phospholipids content, membrane proteins conformation, lipid peroxidation product, and free sulfhydryl of plasma membranes (PMs) interacted with different FFAs were determined. The mammary cells PMs at 38 and 39.5 °C showed different adsorption capacities: acetic acid (Ac) > stearic acid (SA) > β-hydroxybutyric acid (BHBA) > trans10, cis12 CLA. In the FTIR spectrum, the major adsorption peaks appeared at 2920 and 2850 cm^?1 for phospholipids, and at 1628 and 1560 cm^?1 for membrane proteins. The intensities of PMs-FFAs complexes were varied with the FFAs species and their initial concentrations. The β-sheet and turn structures of membrane proteins were transferred into random coil and α-helix after BHBA, SA and trans10, cis12 CLA treatments compared with Ac treatment. The quenching effects on the fluorescence of endogenous membrane protein, 1, 8-ANS, NBD-PE, and DHPE entrapped in PMs by LCFA were different from those of short chain FFAs. These results indicate that the adsorption of FFAs could change membrane protein conformation and polarity of head group in phospholipids. This variation of the mammary cells PMs was regulated by carbon chain length and saturability of FFAs.     

Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzB_SF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzB_SF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process.     

Alkyl Chain Length Effects of Polymer Donors on the Morphology and Device Performance of Polymer Solar Cells with Different Acceptors

The development of nonfullerene acceptors has brought polymer solar cells into a new era. Maximizing the performance of nonfullerene solar cells needs appropriate polymer donors that match with the acceptors in both electrical and morphological properties. So far, the design rationales for polymer donors are mainly borrowed from fullerene‐based solar cells, which are not necessarily applicable to nonfullerene solar cells. In this work, the influence of side chain length of polymer donors based on a set of random terpolymers PTAZ‐TPD10‐Cn on the device performance of polymer solar cells is investigated with three different acceptor materials, i.e., a fullerene acceptor [70]PCBM, a polymer acceptor N2200, and a fused‐ring molecular acceptor ITIC. Shortening the side chains of polymer donors improves the device performance of [70]PCBM‐based devices, but deteriorates the N2200‐ and ITIC‐based devices. Morphology studies unveil that the miscibility between donor and acceptor in blend films depends on the side chain length of polymer donors. Upon shortening the side chains of the polymer donors, the miscibility between the donor and acceptor increases for the [70]PCBM‐based blends, but decreases for the N2200‐ and ITIC‐based blends. These findings provide new guidelines for the development of polymer donors to match with emerging nonfullerene acceptors.     

Creating Order from Chaos: Epigenome Dynamics in Plants with Complex Genomes

Flowering plants have strikingly distinct genomes, although they contain a similar suite of expressed genes. The diversity of genome structures and organization is largely due to variation in transposable elements (TEs) and whole-genome duplication (WGD) events. We review evidence that chromatin modifications and epigenetic regulation are intimately associated with TEs and likely play a role in mediating the effects of WGDs. We hypothesize that the current structure of a genome is the result of various TE bursts and WGDs and it is likely that the silencing mechanisms and the chromatin structure of a genome have been shaped by these events. This suggests that the specific mechanisms targeting chromatin modifications and epigenomic patterns may vary among different species. Many crop species have likely evolved chromatin-based mechanisms to tolerate silenced TEs near actively expressed genes. These interactions of heterochromatin and euchromatin are likely to have important roles in modulating gene expression and variability within species.     

The Localization of Phenolic Compounds in Liposomal Bilayers and Their Effects on Surface Characteristics and Colloidal Stability

The interactions with and effects of five chemically distinct, bioactive phenolic compounds on the lipid bilayers of model dipalmitoylphosphatidylcholine (DPPC) liposomes were investigated. Complementary analytical techniques, including differential scanning calorimetry (DSC) and phosphorus and proton nuclear magnetic resonance spectroscopy (NMR), were employed in order to determine the location of the compounds within the bilayer and to correlate location with their effects on bilayer characteristics and liposomal stability. As compared to the phenolic compounds localized in the glycerol region of the DPPC head group within the bilayer, which enhanced the colloidal stability of the liposomes, compounds located closer to the center of the bilayer reduced vesicle stability as a function of time. Molecules present in the upper region of liposomal DPPC acyl chains (C₁–C₁₀) inhibited liposomal aggregation and size increase, perhaps due to tighter packing of adjoining DPPC molecules and increased surface exposure of DPPC phosphate head groups. These data may be useful for designing liposomal systems containing hydrophobic phenols and other small molecules, selecting appropriate analytical methods for determining their location within liposomal bilayers, and predicting their effects on liposome characteristics early in the liposome formulation development process.