Function Of Biological Membranes Research Articles

Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains

Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization. We have measured tube pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipette-aspirated giant vesicles. We observed that fluorescently-labeled DA-N-BAR showed significantly higher protein density on tubules compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin.

Haematological and Antioxidant Properties of Alloxan-Induced Diabetes Rats Supplemented with Antioxidant Micronutrients

Oxidative stress is elevated in patients with diabetes mellitus and oxidative modification of red cell membrane increases its fragility. Micronutrients with antioxidant effect protect structure and function of biological membranes. In the current study, manganese, copper and zinc, were supplemented in alloxan-induced diabetic rats for a period of 4 weeks. Haematological indices were determined by standard methods and results analyzed using InStat3 Statistical Software. Haemoglobin, packed cell volume, and red blood cell concentrations were 16.91±0.23g/dl, 50.0±1.11% and 5.63±0.32(1012/L) in controls 12.43±0.71g/dl, 36.42±1.38% and 4.58±0.23(1012/L) in treated unsupplemented group, and 13.84±0.43g/dl, 42.14±1.67% and 5.54±0.32(1012/L) in treated and supplemented groups respectively. Mean cell volume, mean cell haematocrit and mean cell haematocrit concentration values were 9.39±0.25fl, 3.15±0.12pg and 0.33±0.01g/L in controls, 8.18±0.46fl, 2.74±0.09pg and 0.34±0.62g/L in the treated unsupplemented group, and 7.99±0.65fl, 2.57±0.18pg and 0.32±0.01g/L in the treated and supplemented group respectively. Activities of superoxide dismutase, glutathione peroxidase and catalase were 2.00±0.17U/mg protein, 46.43±4.25U/mg protein and 59.86±1.08U/mg protein in the controls, 1.67±0.22U/mg protein, 36.88±3.91U/mg protein and 39.86±3.03U/mg protein in the unsupplemented group and 1.61±0.18U/mg protein, 44.86±1.82U/mg protein and 57.14±2.48U/mg protein in the supplemented group respectively. The concentration of malondialdehyde was 1.83±0.16nmol/ml in controls, 2.37±0.19nmol/ml in supplemented group and 1.91±1.14nmol/ml in the supplemented group. It may be concluded from the present study that antioxidant micronutrient improve some haematological and antioxidant indices and reduce lipid peroxidation in alloxan-induced diabetic rats. Keywords: diabetes, haematological indices, micronutrients, lipid peroxidation.

Diffusion in single supported lipid bilayers studied by quasi-elastic neutron scattering

It seems to be increasingly accepted that the diversity and composition of lipids play an important role in the function of biological membranes. A prime example of this is the case of lipid rafts; regions enriched with certain types of lipids which are speculated to be relevant to the proper functioning of membrane embedded proteins. Although the dynamics of membrane systems have been studied for decades, the microscopic dynamics of lipid molecules, even in simple model systems, is still an active topic of debate. Neutron scattering has proven to be an important tool for accessing the relevant nanometre length scale and nano to picosecond time scales, thus providing complimentary information to macroscopic techniques. Despite their potential relevance for the development of functionalized surfaces and biosensors, the study of single supported membranes using neutron scattering poses the challenge of obtaining relevant dynamic information from a sample with minimal material. Using state of the art neutron instrumentation we were, for the first time, able to model lipid diffusion in single supported lipid bilayers. We find that the diffusion coefficient for the single bilayer system is comparable to the multi-lamellar lipid system. More importantly, the molecular mechanism for lipid motion in the single bilayer was found to be a continuous diffusion, rather than the flow-like ballistic motion reported in the stacked membrane system. We observed an enhanced diffusion at the nearest neighbour distance of the lipid molecules. The enhancement and change of character of the diffusion can most likely be attributed to the effect the supporting substrate has on the lipid organization.

Simulations of Lipid Bilayer Domain Formation: Effects of Steroid Structure and Asymmetry

There is a growing amount of evidence that laterally segregated domains of lipids are an integral part of biological membrane structure and function. Using Coarse Grain and Atomistic Molecular Dynamics Simulations we investigate the role of steroid structure and asymmetry in domain formation. Cholesterol, an essential component of animal membranes, appears to be well suited for ordering neighboring lipid chains and promoting domain formation. We demonstrate that alterations to the steroid headgroup hydrophobicity trigger a conversion from domain promoting to domain inhibiting. Those steroids which inhibit domain formation are observed to be less stable in the typical, upright orientation, and instead insert into the bilayer hydrophobic core and reside in an orientation perpendicular to the bilayer normal axis. A second set of simulations are used to address the role of bilayer asymmetry in domain formation. Cellular membranes are thought to be asymmetric, containing different lipid compositions on opposing leaflets, with the outer leaflet capable of domain formation and the inner leaflet uniformly disordered. These simulations suggest how domain formation is affected by an opposing uniformly disordered leaflet.

Quantification of the Nanomechanical Stablility of Multicomponent Lipid Bilayers

Quantification of the mechanical stability of lipid bilayers is important in establishing the composition-structure-property relations, and shed light on understanding functions of biological membranes. We report a direct correlation of the self-organized structures exhibited in phase-segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine/egg sphingomyelin/cholesterol (DEC) in the absence and presence of ceramide (DEC-Ceramide) with their nanomechanical properties using AFM imaging and high-resolution force mapping.

Quantification of the Nanomechanical Stability of Ceramide-Enriched Domains

The quantification of the mechanical stability of lipid bilayers is important in establishing composition-structure-property relations and sheds light on our understanding of the functions of biological membranes. Here, we designed an experiment to directly probe and quantify the nanomechanical stability and rigidity of the ceramide-enriched platforms that play a distinctive role in a variety of cellular processes. Our force mapping results have demonstrated that the ceramide-enriched domains require both methyl beta-cyclodextrin (MbCD) and chloroform treatments to weaken their highly ordered organization, suggesting a lipid packing that is different from that in typical gel states. Our results also show the expulsion of cholesterol from the sphingolipid/cholesterol-enriched domains as a result of ceramide incorporation. This work provides quantitative information on the nanomechanical stability and rigidity of coexisting phase-segregated lipid bilayers with the presence of ceramide-enriched platforms, indicating that that generation of ceramide in cells drastically alters the structural organization and the mechanical property of biological membranes.

Calcium-Dependent Lateral Organization in Phosphatidylinositol 4,5-Bisphosphate (PIP2)- and Cholesterol-Containing Monolayers

Biological membrane function, in part, depends upon the local regulation of lipid composition. The spatial heterogeneity of membrane lipids has been extensively explored in the context of cholesterol and phospholipid acyl-chain-dependent domain formation, but the effects of lipid head groups and soluble factors in lateral lipid organization are less clear. In this contribution, the effects of divalent calcium ions on domain formation in monolayers containing phosphatidylinositol 4,5-bisphosphate (PIP2), a polyanionic, multifunctional lipid of the cytosolic leaflet of the plasma bilayer, are reported. In binary monolayers of PIP2 mixed with zwitterionic lipids, calcium induced a rapid, PIP2-dependent surface pressure drop, with the concomitant formation of laterally segregated, PIP2-rich domains. The effect was dependent upon head-group multivalency, because lowered pH suppressed the surface-pressure effect and domain formation. In accordance with previous observations, inclusion of cholesterol in lipid mixtures induced coexistence of two liquid phases. Phase separation strongly segregated PIP2 to the cholesterol-poor phase, suggesting a role for cholesterol-dependent lipid demixing in regulating PIP2 localization and local concentration. Similar to binary mixtures, subphase calcium induced contraction of ternary cholesterol-containing monolayers; however, in these mixtures, calcium induced an unexpected, PIP2- and multivalency-dependent decrease in the miscibility phase transition surface pressure, resulting in rapid dissolution of the domains. This result emphasizes the likely critical role of subphase factors and lipid head-group specificity in the formation and stability of cholesterol-dependent domains in cellular plasma membranes.

Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.

Antimicrobial peptides: to membranes and beyond

Background: Antimicrobial peptides (AMP) are widely recognized as promising alternatives to the current use of antibiotics and fungicides. Amino-acid sequences of a vast majority of AMP share cationic and amphipathic biophysical properties that allow their insertion into lipid bilayers and can lead to alteration of biological membrane functions. Initial characterization studies linked these properties to antimicrobial killing activity. However, further data indicate that this is not the sole mode of action and that more subtle mechanisms might mediate the interaction with and effect to target microbes, as well as the specificity and toxicity of peptides. As such, AMP are increasingly viewed as powerful multifunctional drugs. Objective: This review summarizes findings on these alternative non-lytic modes of antimicrobial action that go beyond membrane disruption, with an emphasis on the specific interaction with microbial cell wall/membrane components, signaling of AMP exposure and intracellular targets of peptide action. We also explore how novel technologies can help to reveal, characterize and exploit these antimicrobial properties. Conclusion: Detailed knowledge on non-lytic modes of action of AMP will help in the design and discovery of novel antibacterial and antifungal compounds.

Direct Correlation of Structures and Nanomechanical Properties of Multicomponent Lipid Bilayers

Exploring the fine structures and physicochemical properties of physiologically relevant membranes is crucial to understanding biological membrane functions including membrane mechanical stability. We report a direct correlation of the self-organized structures exhibited in phase-segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine/egg sphingomyelin/cholesterol (DEC) in the absence and presence of ceramide (DEC-Ceramide) with their nanomechanical properties using AFM imaging and high-resolution force mapping. Direct incorporation of ceramide into phase-segregated supported lipid bilayers formed ceramide-enriched domains, where the height topography was found to be imaging setpoint dependent. In contrast, liquid ordered domains in both DEC and DEC-Ceramide presented similar heights regardless of AFM imaging settings. Owing to its capability for simultaneous determination of the topology and interaction forces, AFM-based force mapping was used in our study to directly correlate the structures and mechanical responses of different coexisting phases. The intrinsic breakthrough forces, regarded as fingerprints of bilayer stability, along with elastic moduli, adhesion forces, and indentation of the different phases in the bilayers were systematically determined on the nanometer scale, and the results were presented as two-dimensional visual maps using a self-developed code for force curves batch analysis. The mechanical stability and compactness were increased in both liquid ordered domains and fluid disordered phases of DEC-Ceramide, attributed to the influence of ceramide in the organization of the bilayer, as well as to the displacement of cholesterol as a result of the generation of ceramide-enriched domains. The use of AFM force mapping in studying phase segregation of multicomponent lipid membrane systems is a valuable complement to other biophysical techniques such as imaging and spectroscopy, as it provides unprecedented insight into lipid membrane mechanical properties and functions.

Cooperative long range protein-protein dynamics in Purple Membrane

The understanding of dynamics and functioning of biological membranes and in particular of membrane embedded proteins is one of the most fundamental problems and challenges in modern biology and biophysics. In particular the impact of membrane composition and properties and of structure and dynamics of the surrounding hydration water on protein function is an upcoming hot topic, which can be addressed by modern experimental and computational techniques. Very recently, interprotein motions in a carboxymyoglobin protein crystal were reported from a molecular dynamics simulation [Phys. Rev. Lett. 100, 138102 (2008)]. We present experimental evidence for a cooperative long range protein-protein interaction in purple membrane (PM). The dynamics was quantified by measuring the spectrum of the acoustic phonons in the 2d Bacteriorhodopsin (BR) protein lattice using inelastic neutron scattering. The data were compared to an analytical model and the effective spring constant for the interaction between protein trimers was determined to be k=53.49 N/m. The experimental results are in very good agreement to the computer simulations, which reported interaction energy of 1 meV.[1] Maikel C. Rheinstadter, Karin Schmalzl, Kathleen Wood, Dieter Strauch, http://arxiv.org/abs/0803.0959.

Effects Of Hydration On The Dynamics Of Water In Lipid Bilayer Systems: A Molecular Dynamics Study

The properties of interlamellar water are critically important to the structure and function of biological membranes. Recent developments in femtosecond infrared spectroscopy on membrane systems at various hydration levels have opened the possibility of direct comparison between experiment and molecular dynamics simulations on the same time scales. The interpretation of experimental findings can benefit from a detailed computational study of water solvation structure and dynamics of inter-lamellar water at these hydration levels. In this presentation, we report molecular dynamics simulations of 1-palmitoyl-2-oleoyl-phosphatidylcholine POPC bilayers in the liquid-crystalline state and at three hydration levels. Simulations were performed in the canonical ensemble using the GROMACS software package. The extent to which water is influenced by the presence of membrane depends on the hydration level. We found the anisotropic diffusion constant of lipid water exhibits interesting crossover behavior as the water molecule moves from the head group region toward the bulk region. The anisotropic hydrodynamic diffusion of water is explained by structural perturbation of the water hydrogen bond network by the lipid. Radial distribution function, spatial distribution function, and power spectra of water are calculated to consolidate our interpretation. This work was partially supported by the National Science Foundation under Grant No. DGE-0221680.

Single-molecule probes of lipid membrane structure.

Biological membranes are highly heterogeneous structures that are thought to use this heterogeneity to organize and modify the function of membrane constituents. Probing membrane organization, structure, and changes therein are crucial for linking structural metrics with function in biological membranes. Here we report the use of single-molecule fluorescence studies to measure membrane structure at the molecular level. Several groups have shown that polarized total internal reflection fluorescence microscopy using p-polarized excitation can reveal single-molecule orientations when spherical aberrations are introduced into the optics train. We use this approach to measure the orientation of fluorescent lipid analogs doped into Langmuir-Blodgett films of DPPC and arachidic acid. We compare two commonly used fluorescent lipid analogs, BODIPY-PC and DiIC18, which have their fluorophores located in the tailgroup and headgroup, respectively. We find the tilt orientation of BODIPY-PC is very sensitive to the surface pressure at which DPPC films are transferred onto the substrate. At low surface pressures, the tailgroups are largely lying in the plane of the filmand evolve to an orientation normal to the surface as pressure is increased. For DiIC18, however, no evolution in orientation with surface pressure is observed, which is consistent with the headgroup located fluorophore being less sensitive to changes in membrane packing. Single-molecule orientation measurements of DiIC18 in multilayer films of arachidic acid are also measured and compared with previous bulk measurements. Finally, single-molecule measurements are utilized to reveal the ordering induced in DPPC monolayers following the addition of cholesterol.

Molecular Pathology in Atherosclerosis: The Mechanism How Cholesteryl Ester Accumulates in Atheromatous Aorta

To study how cholesterol accumulates in atheroma, novel monoclonal antibodies were developed, using crude homogenate of atheroma as immunogens. 212D monoclonal antibody recognizing extra cellular matrix with lipid-laden deposits was selected by histochemical staining. The antigen was deduced vitronectin from cDNA library. DLH3 monoclonal antibody recognizing oxidized LDL, epitope of which was 5- or 9-phosphatidylcholine. Significant correlations between oxidized LDL and coronary heart disease (CHD) patients were observed from clinical study. 256C monoclonal antibody recognizing atheromatous lesions in human aorta was selected. Epitope must be PC-cholesterol complex which may involve in foam cell rupture. Atherogenesis will be discussed from the aspects of these antibodies. Our working hypothesis is required to elucidate the mechanism. Denatured lipoproteins (either oxidized lipoprotein or ruptured foam cells) may induce atheroma. Oxidation of lipoprotein may be taken place both in foam cells and/or extra cellular matrix, and macrophage eliminate these denatured lipoproteins and become foam cells. The foam cells are ruptured by either apoptosis or necrosis afterward, and hydrophobic fragments of foam cells dispersed in extra cellular space, which destroys the function of biological membrane. Since biological function could be maintained by segregation of hydrophilic circumstances, macrophages uptake these fragmented material and oxidized lipoprotein to maintain the function. This vicious spiral may enhance chronically the atheroma.

Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.

Collective molecular dynamics in proteins and membranes (Review)

The understanding of dynamics and functioning of biological membranes and, in particular, of membrane embedded proteins is one of the most fundamental problems and challenges in modern biology and biophysics. In particular, the impact of membrane composition and properties and of structure and dynamics of the surrounding hydration water on protein function is an upcoming topic, which can be addressed by modern experimental and computational techniques. Correlated molecular motions might play a crucial role for the understanding of, for instance, transport processes and elastic properties, and might be relevant for protein function. Experimentally that involves determining dispersion relations for the different molecular components, i.e., the length scale dependent excitation frequencies and relaxation rates. Only very few experimental techniques can access dynamical properties in biological materials on the nanometer scale, and resolve dynamics of lipid molecules, hydration water molecules, and proteins and the interaction between them. In this context, inelastic neutron scattering turned out to be a very powerful tool to study dynamics and interactions in biomolecular materials up to relevant nanosecond time scales and down to the nanometer length scale. The author reviews and discusses inelastic neutron scattering experiments to study membrane elasticity and protein-protein interactions of membrane embedded proteins.

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function.

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside G M1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.

Using Compression Isotherms of Phospholipid Monolayers To Explore Critical Phenomena. A Biophysical Chemistry Experiment

Two experiments are described in which students explore phase transitions and critical phenomena by obtaining compression isotherms of phospholipid monolayers using a Langmuir trough. Through relatively simple analysis of their data students gain a better understanding of compression isotherms, the application of the Clapeyron equation, the balance between enthalpic and entropic contributions to the chemical potential, critical phenomena, and phase diagrams in general. Students use their data to determine the latent heat of transition, the entropy of transition, and the critical temperature for the liquid condensed to liquid expanded phase transition of a monolayer. Students also gain a general understanding of how molecular level changes in the structure of phospholipids, such as changes in chain length affect the structure and function of biological membranes.

Functional evidence for presence of lipid rafts in erythrocyte membranes: Gsα in rafts is essential for signal transduction

Membrane microdomains enriched in cholesterol and sphingolipids and containing specific membrane proteins are designated as lipid rafts. Lipid rafts have been implicated in cell signaling pathways in various cell types. Heterotrimeric guanine nucleotide-binding protein (Gsalpha) has been shown to be a raft component of erythrocytes and has been implicated in cell signaling. Rafts are isolated as detergent-resistant microdomains (DRMs) for biochemical analysis. Cholesterol depletion is widely used to disrupt raft structures to study their function in biological membranes. In the present study, we developed an alternate strategy for disrupting raft structures without altering membrane cholesterol content. Lidocaine hydrochloride, an amphipathic local anesthetic, is shown to reversibly disrupt rafts in erythrocyte membranes and alter the Gsalpha dependent signal transduction pathway. These findings provide evidence for the presence of rafts while maintaining normal cholesterol content in erythrocyte membranes and confirm a role for raft-associated Gsalpha in signal transduction in erythrocytes.