Top Leaflet Research Articles

Bilayer Asymmetry, Cholesterol Content, and Ligand Binding Influence Membrane Protein Sequestering in Raft-Mimicking Lipid Mixtures

Lipid heterogeneities and membrane proteins are widely considered to show an important functional relationship in plasma membranes. However, the analysis of this intriguing relationship is challenging, due to the small size and transient nature of lipid heterogeneities in the plasma membrane environment. Recently, we presented a powerful model membrane platform that allows the thorough analysis of membrane protein sequestering and oligomerization in well-defined heterogeneous lipid environments using confocal fluorescence intensity analysis paired with a photon counting histogram (PCH) method (1). By applying this experimental approach, here we show that bilayer asymmetry has a significant influence on the sequestering of αvβ3 and α5β1 integrins. Our experiments demonstrate a higher affinity of integrins for the liquid-disordered (ld) phase in the presence of bilayer-spanning liquid-ordered (lo)-ld phase separations. In contrast, a preference for the lo phase is observed in an asymmetric bilayer with lo-ld phase separations, which are exclusively located in the top leaflet of the bilayer. Importantly, PCH analysis shows that the observed changes in integrin sequestering are not caused by altered receptor oligomerization states. In another set of experiments, we also demonstrate that changes in cholesterol may have a profound impact on integrin sequestering without altering receptor oligomerization state. The obtained results are discussed in terms of potential changes in lipid packing density and hydrophobic thickness within the model membrane. The experimental model membrane approach is also applied to explore the functionally important relationship between the sequestering, level of dimerization, and ligand binding of the GPI-anchored urokinase receptor.(1) Siegel, A. P. et al. (2011) Biophys J101(7): 1642-1650.

Leaf Positions of Potato Suitable for Determination of Nitrogen Content with a SPAD Meter

Hand-held SPAD meter can be used to evaluate the leaf nitrogen status of potato. For practical use, it is necessary to select a proper compound leaf, a proper leaflet within compound leaf and position of leaflet suitable for measurement. Therefore, field experiments were conducted in northern China in 2009 and 2010. The SPAD values, plant growth, N uptake of potato plants at tuber initiation and tuber bulking stages under different N supply levels, and final tuber yields were examined. The criteria for determining the most suitable leaf, leaflet and position within a leaflet are that the SPAD values show less variation at a given N supply level, and show a more sensitive response to different nitrogen levels. Our results showed that the coefficients of variance of SPAD values ranged from 8.7 to 25.9% with a leaf N concentration range of 2.1 to 3.8 gN 100 g-1 at tuber initiation stage, and 7.2 to 21.6% with leaf N concentration range of 0.96 to 1.26gN 100 g-1 at the tuber bulking stage. The SPAD values of the 4th compound leaf from apex were more stable and more sensitive to the nitrogen level than those of other leaves, suggesting that the 4th compound leaf is suitable for estimating the leaf N status using a SPAD meter. Within a compound leaf, the SPAD value of the top leaflet was more sensitivethan the other leaflets to nitrogen supply, whereas it was less stable, making it difficult to chose the leaflet for measurement. However, the top leaflet emerges and expands much earlier than the side leaflets, and should be better for SPAD value measurement. The SPAD measurements at the top point of the top leaflet of the 4th leaf demonstrated both less variation and higher sensitivity to nitrogen supply. Therefore, we conclude that the top point of the top leaflet of the 4th compound leaf is the best position for potato N status diagnosis using a SPAD meter.

Interrogating Interfacial Organization in Planar Bilayer Structures

The field of biomimetic planar lipid membranes is finding increased importance as the need to devise sensing systems for biologically important species increases. We approach this area with an eye toward understanding how to interrogate local organization in these complex media. Our primary tools for this purpose are spectroscopy and electrochemistry, where imbedded reporter molecules serve as the information transducers. We use Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) methods to construct planar lipid membranes on hydrophilic solid substrates (Au for electrochemistry, SiO x for spectroscopy). Pyrene tethered to the substrate acts as our probe and AC voltammetry was used to evaluate its redox kinetics, showing slow, distance independent electron transfer between the pyrene moieties and the electrode for both monolayer and bilayer systems. Time-resolved fluorescence data indicate that tethered pyrene resides in a highly rigid environment and that the addition of the top lipid leaflet improves the organization of the bottom lipid leaflet. These data point to the cooperative effect of the bilayer leaflets in creating a system that is comparatively rigid on short length scales, and capable of mediating motion and accessibility of imbedded species.

Polarization Modulation Infrared Reflection−Absorption Spectroscopy Studies of the Influence of Perfluorinated Compounds on the Properties of a Model Biological Membrane

A combination of the Langmuir-Blodgett and Langmuir-Schaefer techniques has been used to build a 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayer at a Au(111) electrode surface with hydrogen-substituted acyl chains in the top leaflet (solution side) and deuterium-substituted acyl chains in the bottom leaflet (gold side). Polarization modulation infrared reflection-absorption spectroscopy was used to determine changes in the conformation and orientation of the acyl chains of DMPC caused by the incorporation of two selected perfluorinated compounds, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), into the top leaflet of the bilayer. The incorporation of perfluorinated compounds into the DMPC bilayer caused a broadening of the methylene peaks and a shift in the methylene band positions toward higher frequencies. In addition, the tilt angle of the acyl chains decreased in comparison to the tilt angle of a pure DMPC bilayer. The reported tilt angles were smaller upon insertion of PFOS ( approximately 24 degrees ) than in the presence of PFOA ( approximately 30 degrees ). Overall, the results show that the incorporation of the perfluorinated acids has an effect on the bilayer similar to that of cholesterol by increasing the membrane fluidity and thickness due to a decrease in the tilt angle of the acyl chains.

Layer-by-Layer PMIRRAS Characterization of DMPC Bilayers Deposited on a Au(111) Electrode Surface

A combination of Langmuir-Blodgett and Langmuir-Schaefer techniques was employed to deposit 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at a gold electrode surface. One leaflet consisted of hydrogen-substituted acyl chains, and the second leaflet was composed of molecules with deuterium-substituted acyl chains. This architecture allowed for layer-by-layer analysis of the structure of the bilayer. Photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to determine the conformation and orientation of the acyl chains of DMPC molecules in the individual leaflets as a function of the potential applied to the gold electrode. The bilayer is adsorbed onto the metal surface when the field applied to the membrane does not exceed approximately 108 V/m. When adsorbed, the bottom leaflet is in contact with a hydrophobic metal surface, and the top leaflet is interacting with the aqueous solution. The asymmetry of the environment has an effect on the orientation of the DMPC molecules in each leaflet. The tilt angle of the acyl chains of the DMPC molecules in the bottom leaflet that is in contact with the gold is approximately 10 degrees smaller than that observed for the top leaflet that is exposed to the solution. These studies provide direct evidence that the structure of a phospholipid bilayer deposited at an electrode surface is affected by interaction with the metal.

A Computational Protocol for the Integration of the Monotopic Protein Prostaglandin H2 Synthase into a Phospholipid Bilayer

Prostaglandin H2 synthase (PGHS) synthesizes PGH 2, a prostaglandin precursor, from arachidonic acid and was the first monotopic enzyme to have its structure experimentally determined. Both isozymes of PGHS are inhibited by nonsteroidal antiinflammatory drugs, an important class of drugs that are the primary means of relieving pain and inflammation. Selectively inhibiting the second isozyme, PGHS-2, minimizes the gastrointestinal side-effects. This had been achieved by the new PGHS-2 selective NSAIDs (i.e., COX-2 inhibitors) but it has been recently suggested that they suffer from additional side-effects. The design of these drugs only made use of static structures from x-ray crystallographic experiments. Investigating the dynamics of both PGHS-1 and PGHS-2 using classical molecular dynamics is expected to generate new insight into the differences in behavior between the isozymes, and therefore may allow improved PGHS-2 selective inhibitors to be designed. We describe a molecular dynamics protocol that integrates PGHS monomers into phospholipid bilayers, thereby producing in silico atomistic models of the PGHS system. Our protocol exploits the vacuum created beneath the protein when several lipids are removed from the top leaflet of the bilayer. The protein integrates into the bilayer during the first 5 ns in a repeatable process. The integrated PGHS monomer is stable and forms multiple hydrogen bonds between the phosphate groups of the lipids and conserved basic residues (Arg, Lys) on the protein. These interactions stabilize the system and are similar to interactions observed for transmembrane proteins.

Computational Biology

Computational Biology

Probing the Structure of Supported Membranes and Tethered Oligonucleotides by Fluorescence Interference Contrast Microscopy

Fluorescence interference contrast microscopy (FLIC) is a powerful method to structurally characterize fluorescent objects with nanometer-scale resolution in the z direction. Here we use FLIC to characterize the water layer underlying supported membranes and membrane-tethered double-stranded oligonucleotides. FLIC measurements of supported membranes containing the lipid-anchored fluorescent dye DiI in both leaflets indicate the thickness of the water layer separating the solid support and the lower lipid leaflet is 1.3 +/- 0.2 nm. Addition of cobalt(II) chloride to a DiI-supported membrane quenches the fluorescence in the top leaflet of the supported membrane; FLIC measurements of this system precisely locate the DiI to the bottom leaflet. These experiments confirm the accuracy of the model and parameters used to determine the water layer thickness, demonstrate the ability to differentiate between fluorescent objects whose average position differs by approximately 1.9 nm, and provide a widely applicable method to test the resolution of other high-z-resolution fluorescent microscopies. FLIC measurements of Alexa-labeled double-stranded oligonucleotides tethered to a supported membrane indicate that the DNA double helix is oriented perpendicular to the surface. Complications that arise from uncertainly in the orientation of the fluorophore are discussed. Several improvements in FLIC methodology are described. These stringent tests of the resolution of FLIC and the ability to unambiguously determine fluorescent lipid distribution provide structural insight on assemblies at membrane interfaces and permit the detection of even subtle changes at such interfaces.