Bottom Leaflet Research Articles

A Small Molecule Impedes the Aβ1-42 Tetramer Neurotoxicity by Preserving Membrane Integrity: Microsecond Multiscale Simulations.

Amyloid-β (Aβ1-42) peptides aggregated into plaques deposited in the brain are the main hallmark of Alzheimer's disease (AD), a social and economic burden worldwide. In this context, insoluble Aβ1-42 fibrils are the main components of plaques. The recent trials that used approved AD drugs show that they can remove the fibrils from AD patients' brains, but they did not halt the course of the disease. Mounting evidence envisages that the soluble Aβ1-42 oligomers' interactions with the neuronal membrane trigger higher cell death than Aβ1-42 fibril interactions. Developing a compound that can alleviate the oligomer's toxicity is one of the most demanding tasks for curing the disease. We performed two molecular dynamics (MD) simulations in an explicit solvent model. In the first case, 55-μs of multiscale all-atom (AA)/coarse-grained (CG) MD simulations were carried out to decipher the impact of a previously described small anti-Aβ molecule, termed M30 (2-octahydroisoquinolin-2(1H)-ylethanamine), on an Aβ1-42 tetramer structure in close contact with a DMPC bilayer. In the second case, 15-μs AA/CG MD simulations were performed to rationalize the dynamics between Aβ1-42 and Aβ1-42-M30 tetramer complexes embedded in DMPC. On the membrane bilayer, we found that the Aβ1-42 tetramer penetrates the bilayer surface due to unrestricted conformational flexibility and many contacts with the membrane phosphate groups. In contrast, no Aβ1-42-M30 tetramer penetration was observed during the entire course of the simulation. In the case of the membrane-embedded Aβ1-42 tetramer, the integrity of the bottom bilayer leaflet was severely affected by the interactions between the negatively charged phosphate groups and the positively charged residues of the Aβ1-42 tetramer, resulting in a deep tetramer penetration into the bilayer hydrophobic region. These contacts were not observed in the case of the membrane-embedded Aβ1-42-M30 tetramer. It was noted that M30 molecules bind to Aβ1-42 tetramer through hydrogen bonds, resulting in a conformational stable Aβ1-42-M30 complex. The associated complex has reduced conformational changes and an enhanced rigidity that prevents the tetramer dissociation by interfering with the tetramer-membrane contacts. Our findings suggest that the M30 molecules could bind to Aβ1-42 tetramer resulting in a rigid structure, and that such complexes do not significantly perturb the membrane bilayer organization. These observations support the in vitro and in vivo experimental evidence that the M30 molecules prevent synaptotocity, improving AD-affected mice memory.

Self-Uptake Mechanism of Polymyxin-Based Lipopeptide against Gram-Negative Bacterial Membrane: Role of the First Adsorbed Lipopeptide.

Research in the continuously increasing threat of polymyxin-resistant multidrug-resistant Pseudomonas aeruginosa, which causes severe infection in immunocompromised patients, has resulted in the development of several polymyxin-derived cyclic lipopeptides containing l-α-γ- diamino butyric acid-like FADDI-019 (F19). In this work, F19's insertion into a minimal model of the asymmetric outer membrane of the bacterium, which contained only penta-acylated lipid A (LipA) and lacked keto-d-octulosonic acid and O-antigens, in the top leaflet and phospholipids in the bottom leaflet, was studied. F19 exhibited all of the hallmarks of the self-uptake mechanism into the asymmetric bilayer. While a single monomer of the lipopeptide did not get partitioned into the inside of the bilayer, it competitively displaced Ca2+ from the membrane surface, observed as a decrease in Ca2+ coordination number with phosphate groups (1.89 vs 1.718), resulting in membrane destabilization. This resulted in an increment of the average defect size and the probability of interplay between lipid tails and hydrophobic residues of another F19. When more than one monomer was present in the system, the first monomer remained docked on the surface, while other monomers intercalated into the bilayer interior with their hydrophobic moieties "sleeved" by lipid acyl chains. The free energy barrier for partial insertion of the lipopeptide into a bilayer in the presence of surface-docked second F19 was recorded at ∼1.3 kcal/mol using two-dimensional (2D) well-tempered metadynamics, making it a low barrier process at 300 K. This study is an attempt to demonstrate the self-uptake mechanism of F19 during intercalation process into the bilayer interior, which may help in the design of better alternates for polymyxins to work against polymyxin resistance.

Systematic Measurements of Interleaflet Friction in Supported Lipid Bilayers

Along with membrane viscosity, interleaflet friction determines the dynamics of membrane deformation. The two leaflets that form a lipid membrane must slide past each other when the membrane bends, as occurs when vesicles are formed, or membrane tethers are pulled. Existing measurements of interleaflet friction are sparse, examining a single type of lipid, dye, and substrate, which does not give any conclusive answers to how changing these factors will affect the measured interleaflet friction. A recently developed method applies high shear stresses to supported lipid bilayers formed in microfluidic channels, resulting in a tank treading motion where the upper leaflet of the bilayer slides overtop of the bottom leaflet.

Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP2 molecules in the top leaflet are fully mobile, while the PIP2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H2O to D2O, it was shown that the primary interaction between PIP2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP2. Moreover, PIP2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

Role of Pore Forming Toxins in Modulating the Lipid Dynamics

Pore forming toxins (PFT's) are important bacterial virulence factors secreted by bacteria and pathogens. These are water-soluble monomers that spontaneously oligomerize and form self-assembled structures on the host membrane cell, intake nutrition, inhibit the activity ultimately causing cell lysis and cell death. In this study, we investigate the influence of PFT's on the dynamics of two mammalian cell membranes (DMPC and DOPC) in the presence of cholesterol. We show that cholesterol fluidizes the membrane accelerating the dynamics. In the absence of cholesterol, the lipids form crystallized lipid-protein complexes causing a slowdown in dynamics in the vicinity of the protein. We also found that the dynamics of lipids is strongly coupled to their local structure and also on the nature of lipid-protein interactions. The lipid-protein interactions are influenced by the dominant structure of the transmembrane protein in the lipid bilayer and hence depends on the type of the PFT. There exists structural and dynamical heterogeneities in the lipid bilayer and in each leaflet very similar to fluids approaching glass transition. We show that the upper leaflet from where the toxin inserts into the membrane exhibits a slower dynamics than the bottom leaflet and the alpha-PFT perturbs the membrane to a greater extent than the beta-PFT. This study will shed light on the toxicity level of the PFT in influencing the lipid dynamics and the role of cholesterol on lipid-protein interactions.

Molecular Dynamics Simulations of 38 Types of Ganglioside in Homoegneous Membrane Bilayers

Ganglioside is a membrane lipid in which the hydrophilic head group is a complex oligosaccharide containing one or more sialic acids. The oligosaccharides of the ganglioside extend out from the cell surface, interacting with various signaling molecules. Some of their functions include regulation of protein activities, recognition of specific molecules, and communication between cells. Gangliosides are especially abundant in the brain, with three times as much in grey as in white matter. It is believed that gangliosides are involved in pathological states such as cancer, Tay-Sachs disease, Huntington's disease, Alzheimer's disease, et certera. To explore and understand the orientation, structure, and dynamics of gangliosides, molecular dynamics simulations on 38 different types of gangliosides in homogenous membrane bilayer (POPC) were performed. CHARMM-GUI was utilized to build the membrane bilayers, and a ganglioside was inserted in both the upper leaflet and the bottom leaflet, with three simulations for each ganglioside. Each system was ran for up to 50 ns and multiple analyses were ran. Through comparative analysis, the difference in properties and dynamics due to different glycan sequence will be studied. Future work includes running the simulation longer up to 200∼300 ns and assembling the information gathered through multiple analyses for greater understanding of gangliosides, which will aid in glycome studies.

Molecular dynamics simulations reveal how characteristics of surface and permeant affect permeation events at the surface of soft matter

Molecular dynamics simulations are particularly useful in providing details that permit understanding of phenomena occurring at surfaces, phenomena characterised by surface-sensitive experimental methods yielding average properties. The methods and examples that we consider here reveal how the characteristics of the surface and the permeant affect permeation events at the surface of soft matter, in particular, lipid bilayers. We choose permeation of lipid membranes as our example of the ability of molecular dynamics simulations to provide molecular-level mechanisms that are otherwise not available via other means. Molecular permeation through lipid membranes is a fundamental biological process that is important for small molecules such as therapeutics as well as nanoparticles that have become important modes of drug delivery. We describe methods applicable to such large systems and use examples where the permeants are uncharged particles: small molecules (Xe, O2, CO2), bare gold nanocrystals, gold-core nanoparticles with hydrophobic ligands (alkane thiols of various lengths) and gold-core nanoparticles with hydrophilic ligands (methyl-terminated polyethylene glycol of various lengths). In each example, we have previously validated our findings by comparisons with experimental data, using such information as is available from X-ray diffraction, electron paramagnetic resonance, nuclear magnetic resonance, atomic force microscopy, various imaging methods, diffusion measurements, dynamic light scattering. In addition to spherical core nanoparticles, we also examine the characteristic permeation mechanisms of gold nanorods with polyethylene glycol ligands, where the aspect ratio different from 1 makes the permeation event dependent on the angle of the rod axis relative to the membrane surface. This review examines the phenomena associated with the interaction of various permeants with the lipid bilayer that serves as our model membrane. We consider adsorption at the interface, the permeant within the top lipid leaflet, in the middle of the membrane within the lipid tail region, within the bottom lipid leaflet and finally exiting the membrane on the way to recovery, for various permeants: gas molecules, bare gold nanocrystal, gold nanoparticles with alkane thiol ligands, PEGylated gold nanoparticles and PEGylated gold nanorods. We observe formation of a water pore, occasional transport of ions, lipid flip-flops, lipid displacement from the membrane, and rotational behaviour of PEGylated nanorods during the permeation process. These events differ depending on the chemical nature (hydrophobic or hydrophilic) of the ligands, their length, the coverage density on the gold surface and the aspect ratio of the gold core. Direct comparisons across the board are possible by using identical interaction models (MARTINI coarse grain), molecular dynamics methods, simulation set-ups and analyses of MD results, thereby permitting generalisations to be made about mechanisms for the various events and how they are affected by these factors.

Micropatterning of Phase-Segregated Supported Lipid Bilayers and Binary Lipid Phases through Polymer Stencil Lift-Off.

Supported lipid bilayers (SLBs) provide an excellent model system for studying structural and functional characteristics of biomembranes. Patterning model membranes on solid supports has elicited much interest because lipid bilayer arrays at cellular or subcellular scales provide attractive platforms for reconstituting tissue-like conditions for cell culture, and for creating simplified physiological environments to study biological processes. Phase-segregated SLB patterns can be especially useful for such studies, as the selective functionalization of the lipid phases with different lipids, receptors, or proteins can be achieved to mimic the key features of plasma membrane. However, it remains challenging to pattern phase-segregated lipid bilayers and to spatially control the lipid phases at the micron scale. Current methods to achieve this involve multiple surface modification and patterning steps, elaborate techniques such as microfluidic, microcontact printing, or electrochemical control, among others. To overcome the complexity in producing phase-segregated patterns, we have developed simple and rapid strategies to pattern SLBs with phase separation utilizing the polymer stencil lift-off (PSLO) technique. PSLO is a powerful technique for SLB patterning, since it allows the faithful pattern transfer of micron-sized lipid domains onto solid surfaces under aqueous conditions, which eliminates the need for controlled humidity and reduces the risk of bilayer disruption through drying. By integrating postetching substrate cleaning and a blocking treatment, well-defined homogeneous and phase-segregated SLB patterns were achieved with lipid mobility that matches that of SLBs formed on clean SiO2 wafer substrates. A two-step incubation method was also developed for patterning binary lipid phases, which allowed precise control of their position and geometries. The created phase-segregated SLB patterns were used to study lipid phase behavior within confined areas, and quantitative analysis showed that smaller pattern sizes resulted in smaller gel phase domains, which also covered a smaller fraction of the total patterned SLB area. This was attributed to the decreased mobility of the bottom leaflet of the SLB, which lies in close proximity to the substrate, and the resulting hindered exchange of lipid molecules between the bottom and upper leaflets through the SLB boundary. By further integration with functional groups, the phase-segregated lipid bilayer patterns might find relevant application in tissue engineering, biophysical studies of biomolecular and cellular interactions, and biosensing platforms.

Molecular Dynamics Simulations of 41 Types of Ganglioside in Membrane Bilayers

Ganglioside is a membrane lipid in which the hydrophilic head group is a complex oligosaccharide containing one or more sialic acids. The oligosaccharides of the ganglioside extend out from the cell surface, interacting with various signaling molecules. Some of their functions include regulation of protein activities, recognition of specific molecules, and communication between cells. Gangliosides are especially abundant in the brain, with three times as much in grey as in white matter. It is believed that gangliosides are involved in pathological states such as cancer, Tay-Sachs disease, Huntington's disease, Alzheimer's disease, et certera. To explore and understand the orientation, structure, and dynamics of gangliosides, molecular dynamics simulations on 41 different types of gangliosides in homogenous membrane bilayer were performed. CHARMM-GUI was utilized to build the membrane bilayers, and a ganglioside was inserted in both the upper leaflet and the bottom leaflet, with three simulations for each ganglioside. Future work includes assembling the information gathered through multiple analyses for greater understanding of gangliosides, which will aid in glycome studies.

Phase transition behaviors of the supported DPPC bilayer investigated by sum frequency generation (SFG) vibrational spectroscopy and atomic force microscopy (AFM).

The phase transition behaviors of a supported bilayer of dipalmitoylphosphatidyl-choline (DPPC) have been systematically evaluated by in situ sum frequency generation (SFG) vibrational spectroscopy and atomic force microscopy (AFM). By using an asymmetric bilayer composed of per-deuterated and per-protonated monolayers, i.e., DPPC-d75/DPPC and a symmetric bilayer of DPPC/DPPC, we were able to probe the molecular structural changes during the phase transition process of the lipid bilayer by SFG spectroscopy. It was found that the DPPC bilayer is sequentially melted from the top (adjacent to the solution) to bottom leaflet (adjacent to the substrate) over a wide temperature range. The conformational ordering of the supported bilayer does not decrease (even slightly increases) during the phase transition process. The conformational defects in the bilayer can be removed after the complete melting process. The phase transition enthalpy for the bottom leaflet was found to be approximately three times greater than that for the top leaflet, indicating a strong interaction of the lipids with the substrate. The present SFG and AFM observations revealed similar temperature dependent profiles. Based on these results, the temperature-induced structural changes in the supported lipid bilayer during its phase transition process are discussed in comparison with previous studies.

Modeling and Simulations of Glycosphingolipids Determining A, B, and O Blood Groups

Glycosphingolipids are the biological recognition sites in determining human A, B, and O blood groups. They are composed of the long-chain amino alcohol sphingosine, a long-chain fatty acid, and an oligosaccharide polar head group that is joined by a glyosidic linkage. These oligosaccharide sequences that determine human A, B, and O blood groups are almost identical except one terminal sugar (galactose). To understand the structural function and dynamics of the A, B, and H glycoshingolipids and how they act as receptors on the red blood cell to determine the A, B, and O blood groups, we performed molecular dynamics simulations for all three blood type antigens in a membrane bilayer environment. Using CHARMM-GUI (www.charmm-gui.org), we built a membrane bilayer and put one Blood Type A antigen in the upper leaflet and one in the bottom leaflet, and simulated it three times. This was done for Blood Type B antigen and Blood Type H antigen. Future work involves gaining a deeper understanding of the glycosphingolipids’ structure and dynamics, which will help lead to a better functional understanding of the human glycome.

Effect of Asymmetry on Bilayer Membrane Systems

Many biological processes, such as membrane fusion and the action of microbial proteins, involve asymmetric uptake of proteins. In addition, it has been demonstrated that large mechaosensitive channels open by the asymmetric inclusion of lyso-lipids in the bilayer in the absence of external pressure. These phenomena can be interpreted in terms of the asymmetric local pressure profile in the bilayer, which lacks the quantitative understanding. By noting that such asymmetric pressure profile can be induced in bilayer membranes with asymmetric number of lipids between leaflets, to provide quantitative understanding, we performed molecular dynamics simulations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes for a set of systems with different level of asymmetry with and without gramicidin A (gA) or WALP23. We observed that even highly asymmetric lipid bilayers with and without gA or WALP23 were stable during the whole simulation time, which is consistent with the previous study of dipalmitoyl-phosphatidylcholine (DPPC) bilayers [J. Am. Chem. Soc. 131, 15194 (2009)]. As the number of lipids in the lower leaflet becomes smaller, the lipids are more tightly packed in the top leaflet and less ordered in the bottom leaflet, which are quantified in terms the lateral pressure profile, the order parameter, the area per lipid, etc. However, the hydrophobic thickness for pure lipid bilayers does not show meaningful dependence on the asymmetry, which results from the stretched and less ordered lipids in the upper and lower leaflets, respectively. We will also discuss the influence of the asymmetry on the properties of protein-lipid interactions, such as the distance dependence of the lipid properties and the interaction patterns around gA or WALP23.

Membrane attachment and structure models of lipid storage droplet protein 1

Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center.

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

Molecular dynamics simulations of DPPC bilayers using "LIME", a new coarse-grained model.

A new intermediate resolution model for phospholipids, LIME, designed for use with discontinuous molecular dynamics (DMD) simulations is presented. The implicit-solvent model was developed using a multiscale modeling approach in which the geometric and energetic parameters are obtained by collecting data from atomistic simulations of a system composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) molecules and explicit water. In the model, 14 coarse-grained sites that are classified as 1 of 6 types represent DPPC. DMD simulations performed on a random solution of DPPC resulted in the formation of a defect-free bilayer in less than 4 h. The bilayer formed quantitatively reproduces the main structural properties (e.g., area per lipid, bilayer thickness, bond order parameters) that are observed experimentally. In addition, the bilayer transitions from a liquid-crystalline phase to a tilted gel phase when the temperature is reduced. Transbilayer movement of a lipid from the bottom leaflet to the top leaflet is observed when the temperature is increased.

Length and Sequence Dependence in the Association of Htt Protein with Lipid Bilayers

There is a fundamental gap in our understanding of how aggregates formed by mutant Huntington (htt) protein with varying polyQ repeats gain toxic properties causing Huntington's disease (HD). From experimental studies, it has been known that the central and the most important step associated with toxicity is the binding of mutant htt and its aggregates to lipids. It is also known that flanking amino acid sequences around the polyglutamine stretch directly affects the interaction with the lipid bilayer. The exact nature of these interactions and the key steps of how these flanking sequences and polyglutamate length affect structure of lipid bilayers are not known. In this study, we use atomistic molecular dynamics simulations to study the nature of interaction between the various mutant htt protein with the associated flanking sequences around the polyQ stretch and we explore the changes in these interactions with lipid bilayers by varying the length of the polyQ stretch as well. We also study the effect of lipid compositions on these interactions. Initial results from adsorption simulations of polyQ peptides show membrane thinning and changes in the area of lipid head groups where the protein adsorbs. Although we do not observe complete disruption of membrane structure, we see significant compensation effects in the bottom leaflet of the membrane. We compare these effects across the different flanking sequences and various lengths of polyQ stretches and observe varying degrees of changes in the structure of the lipid bilayer.

Integrin Sequestering in Raft-Mimicking Lipid Mixtures: The Role of Bilayer Asymmetry and Cholesterol Content

It is widely accepted that lipid microdomains play an important functional role in plasma membranes. However, small size and transient nature of lipid/membrane heterogeneities in the plasma membrane make a characterization of microdomains and microdomain-related membrane processes in cellular systems quite challenging. To address this important problem, we recently introduced a powerful model membrane system that allows the investigation of membrane protein sequestering and oligomerization in raft-mimicking lipid mixtures using combined confocal fluorescence spectroscopy, photon counting histogram (PCH), and epifluorescence microscopy (1). Our experiments on bilayer-spanning domains showed that αvβ3 and α5β1 integrins predominantly exist as monomers and sequester preferentially to the liquid-disordered (ld) phase in the absence of ligands. Notably, addition of vitronectin (αvβ3) and fibronectin (α5β1) caused substantial translocations of integrins into the liquid-ordered (lo) phase without altering receptor oligomerization state. Here we expand our previous studies and report on the sequestering and oligomerization state of αvβ3 and α5β1 in asymmetric bilayer compositions containing coexisting lo and ld phases located exclusively in the top leaflet of the bilayer (bottom leaflet shows only ld phase). Remarkably, in such a membrane environment, both integrins show a higher affinity for the top leaflet-restricted lo domains in the absence of their respective ligands. This sequestering behavior of integrins was only slightly modified after addition of their respective native ligands. Our findings show that cholesterol content has a substantial influence on integrin sequestering and oligomerization in raft-mimicking lipid mixtures. The described experimental results highlight the potential importance of membrane asymmetry and lipid composition in the sequestering of membrane proteins in biological membranes.(1) Siegel, A. P. et al. (2011) Biophys J 101(7): 1642-1650.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A2 (PLA2) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA2 toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA2 and most of the hydrolysis products desorb from substrate surface in 40min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA2 increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA2 preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA2-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA2 mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA2.

Making A Permanent Membrane Raft fromTethered Cholesterol

It is well established that the presence of cholesterol increases the stability and rigidity of lipsomes by increasing their area-expansion modulus and bending energies. In nature, cholesterol molecules in the cell membrane are known to phase separate into cholesterol rich and cholesterol deficient domains, leading to the formation of “rafts”. Here, we demonstrate the creation of a permanent raft, i.e., a robust supported lipid bilayer, using immobilized and dispersed cholesteryl groups covalently anchored to a hydrophilic polymer brush. This allows a uniform interaction of cholesteryl groups with the entire bottom leaflet of an supported lipid bialyer (SLB). When the surface cholesteryl concentration is 0.3 per square nanometer or higher, we obtain an air stable SLB while maintaining fluidity of the lipid membrane environment. The fluidic and air-stable SLB is not only a robust model for biophysical studies of membranes, but also an efficient cell-mimicking platform for high throughput analysis.