Proton transport through a peptide-tethered bilayer lipid membrane by the H(+)-ATP synthase from chloroplasts measured by impedance spectroscopy.

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.     

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, III: Molecular Dynamics Simulation Incorporating a Cyanophenylalanine Spectroscopic Probe

A nitrile-derived amino acid, Phe_CN, has been used as an internal spectroscopic probe to study the binding of an inhalational anesthetic to a model membrane protein. The infrared spectra from experiment showed a blue-shift of the nitrile vibrational frequency in the presence of the anesthetic halothane. To interpret the infrared results and explore the nature of the interaction between halothane and the model protein, all-atom molecular dynamics (MD) simulations have been used to probe the structural and dynamic properties of the protein in the presence and absence of one halothane molecule. The frequency shift analyzed from MD simulations agrees well with the experimental infrared results. Decomposition of the forces acting on the nitrile probes demonstrates an indirect impact on the probes from halothane, namely a change of the protein's electrostatic local environment around the probes induced by halothane. Although the halothane remains localized within the designed hydrophobic binding cavity, it undergoes a significant amount of translational and rotational motion, modulated by the interaction of the trifluorine end of halothane with backbone hydrogens of the residues forming the cavity. This dominant interaction between halothane and backbone hydrogens outweighs the direct interaction between halothane and the nitrile groups, making it a good “spectator” probe of the halothane-protein interaction. These MD simulations provide insight into action of anesthetic molecules on the model membrane protein, and also support the further development of nitrile-labeled amino acids as spectroscopic probes within the designed binding cavity.     

A giant liposome for single-molecule observation of conformational changes in membrane proteins

We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (F_OF₁) in liposomes tens of μm in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead.     

Liposomes as a Model for the Biological Membrane: Studies on Daunorubicin Bilayer Interaction

In this study the interaction of the antitumoral drug daunorubicin with egg phosphatidylcholine (EPC) liposomes, used as a cell membrane model, was quantified by determination of the partition coefficient (K _p). The liposome/aqueous-phase K _p of daunorubicin was determined by derivative spectrophotometry and measurement of the zeta-potential. Mathematical models were used to fit the experimental data, enabling determination of K _p. In the partition of daunorubicin within the membrane both superficial electrostatic and inner hydrophobic interactions seem to be involved. The results are affected by the two types of interaction since spectrophotometry measures mainly hydrophobic interactions, while zeta-potential is affected by both interpenetration of amphiphilic charged molecules in the bilayer and superficial electrostatic interaction. Moreover, the degree of the partition of daunorubicin with the membrane changes with the drug concentration, due mainly to saturation factors. Derivative spectrophotometry and zeta-potential variation results, together with the broad range of concentrations studied, revealed the different types of interactions involved. The mathematical formalism applied also allowed quantification of the number of lipid molecules associated with one drug molecule.     

Evidence for nucleotide-dependent passive H+ transport protein in the plasma membrane of barley roots

Plasma membranes were isolated from barley roots by two-phase partitioning, and octylglucoside-soluble and -insoluble fractions were obtained. The insoluble fractions were reconstituted into liposomes, and the plasma membrane H(+)-ATPase was shown to participate in MgATP-dependent H(+) transport activity. The H(+) transport was decreased when the octylglucoside-soluble fraction was reconstituted together with the insoluble fraction. The decrease was not due to inhibition of the H(+)-ATPase, but rather was likely due to the increased H(+) leakage from the proteoliposome. The octylglucoside-soluble fraction was, therefore, reconstituted in the liposomes and the passive H(+) transport was determined using the pH jump method. A pH gradient across the membranes was generated by the pH jump, and the gradient was found to be dissipated by passive H(+) transport. The H(+) transport required ATP, K(+), and valinomycin. The H(+)-transport also occurred when ADP, AMP, GTP, or ATP-gamma-S was present instead of ATP, and did not occur when the octylglucoside-soluble fraction was boiled before the reconstitution. These findings suggest that nucleotide-dependent H(+ )transport protein is present in the plasma membrane of root cells.     

Determination of membrane potential with lipophilic cations. Comparison of estimated values with various phosphonium ions

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–5) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the binding of probes to the membrane was measured. For the probes of n = 0 and n = 1, and for TPP⁺, binding followed the Langmuir adsorption isotherm. For other probes, analysis revealed the presence of two, high- and low-affinity, binding sites. Upon illumination, which generated the membrane potential, the probe molecules were accumulated into the vesicles. If we ignore the membrane-potential-dependent binding of the probe molecules, the estimated values are larger when the probe used is more hydrophobic. We have tested some models describing the amount of probe bound on membranes in terms of concentration of free probe inside and outside the vesicles. No model has fulfilled the criterion of valid estimation that the membrane potentials estimated are independent of probes used. An experimental method for the estimation of true membrane potential is proposed. Effects of tetraphenylboron on the estimation of membrane potential and on the transport rate of phosphonium cations were examined.     

Structural changes of liposome phospholipid packing induced by cytotoxin of the Central Asia cobra venom

Liposomes and proteoliposomes obtained from rat brain were used; structural changes induced by Vc5 cytotoxin (CT) from Central Asia cobra venom have been studied by the EPR method using spin probes (5-, 10-, or 12-doxylstearic acid). The addition of CT to liposome samples, containing spin probes resulted in the appearance of a new EPR signal in the initial spectrum (samples without CT), typical of probes with strongly retarded mobility. The presence of hydrophobic interaction between the CT molecules and spin labelled fat acids permits the assumption that CT molecules in liposomes trap both lipid probes and phospholipids localized in the reach of action of hydrophobic forces. CT may be supposed to induce formation in membranes of liposomes with domain structures. As a result of hydrophobic interaction with CT molecules both the phospholipid and lipid probe mobility in the domain is substantially less than that in liposome regions free of CT molecules. Due to this, a new signal appears in the initial EPR spectrum of the spin probes. An analysis of the dependence of the probe order parameter value on CT concentration in samples has suggested that CT act uniformly along the membrane lipid profile with a certain CT concentration range. At high concentrations CT molecules cannot penetrate the lipid region deep enough, due to mutual electrostatic repulsion and steric factors at membrane surface. As a result, structural changes involve regions adjacent to the membrane surface only.     

Mean residence time of molecules diffusing in a cell bounded by a semi-permeable membrane: Monte Carlo simulations and an expression relating membrane transition probability to permeability

 The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn²⁺ and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P _d. This relationship was derived and used to show that the mean residence time for a water molecule is determined by P _d when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value. Received: 7 January 2000 / Revised version: 4 April 2000 / Accepted: 4 April 2000     

Effect of cholesterol on molecular transport of organic cations across liposome bilayers probed by second harmonic generation

The effect of cholesterol on the molecular transport of an organic cation, malachite green (MG), across large unilamellar dioleolyphosphatidylglycerol (DOPG) liposome bilayers with 0-50 mol% cholesterol was studied by second harmonic generation (SHG). Because SHG is a surface-specific technique, it requires no labeled molecule, quencher, or shifting agent to distinguish the location of the solute molecules. An additional important feature of SHG is that it is sensitive only to the probe molecules bound to the liposome, whereas other methods can only differentiate between molecules that are outside and those inside the liposome. The transport kinetics of MG across the liposome bilayers was observed in real time, and the results show that cholesterol retards the rate of transport of MG across liposome bilayers. The rate was found to decrease by six times for 50 mol% cholesterol content compared with cholesterol-free liposomes. This demonstrates the applicability of SHG to investigation of the effect of liposome composition on the transport kinetics across the liposome bilayers.     

HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z_HER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as “Affisomes” were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80–100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41°C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16–20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4–12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were?～70% at a protein-lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm?～0°C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained?>?90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.     

Effect of egg‐phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa

This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling. Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5‐carboxyfluorescein diacetate (cFDA)) at 16°C, 12°C, 4°C, and 22°C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope. The LPT center, T_m, was determined by statistical analysis. The LPT and T_m were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg‐phosphatidylcholine (EPC) liposomes at 16°C, 12°C, 4°C, and 26°C (control). The results show that the membrane integrity of spermatozoa incubated at 16°C, 12°C, and 4°C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26°C) and 10°C, with T_m at 14–16°C. The T_m for sperm incubated with liposomes or egg‐yolk extender was below the measured range (2°C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated‐to‐polyunsaturated FAs. Here we show that the protection afforded by the presence of egg yolk or liposomes in the extender is accomplished by shifting the T_m to below the 4°C point at which chilled semen is maintained for transport, or the point at which fast freezing begins to minimize cellular damage. Zoo Biol 0:1–13, 2005. © 2005 Wiley‐Liss, Inc.     

Water permeability of plant cuticles: permeance,diffusion and partition coefficients

Summary Using isolated cuticular membranes from ten woody and herbaceous plant species, permeance and diffusion coefficients for water were measured, and partition coefficients were calculated. The cuticular membranes of fruit had much higher permeance and diffusion coefficients than leaf cuticular membranes from either trees or herbs. Both diffusion and partition coefficients increased with increasing membrane thickness. Thin cuticles, therefore, tend to be better and more efficient water barriers than thick cuticles. We compared the diffusion coefficients and the water content of cuticles as calculated from transport measurements with those obtained from water vapor sorption. There is good to fair agreement for cuticular membranes with a low water content, but large discrepancies appear for polymer matrix membranes with high permeance. This is probably due to the fact that diffusion coefficients obtained from transport measurements on membranes with high permeance and water content are underestimated. Water permeabilities of polyethylene and polypropylene membranes are similar to those of leaf cuticular membranes. However, leaf cuticles have much lower diffusion coefficients and a much greater water content than these synthetic polymers. This suggests that cuticles are primarily mobility barriers as far as water transport is concerned.     

Reconstitution in liposomes of the electron-transport chain catalyzing fumarate reduction by formate

Fumarate reduction by formate in Vibrio succinogenes is catalyzed by a membrane-bound electron-transport chain, and is coupled with the phosphorylation of ADP. The electron-transport chain was reconstituted in liposomes from the isolated components. The formate dehydrogenase complex (three different peptides), the fumarate reductase complex (three different peptides) and vitamin K-1 were required for the electron transport. The pathway of the electrons from formate to fumarate in the reconstituted chain was identical with that in the bacterial membrane. Each of the active enzyme complexes in the liposomes participated in the electron transport. This was valid for proteoliposomes with ratios of the contents of the two enzyme complexes ranging between 0.1 and 10. This indicates that vitamin K-1 forms a diffusible pool within the liposomal membrane that allows every quinone molecule to react with each molecule of the two enzyme complexes.     

Interaction of alcohols with the transport system of glucose in human erythrocytes

Alcohols inhibit the exchange transport of glucose in human erythrocytes. Comparing the inhibition by monohydroxy-alcohols, which have different distribution coefficients between medium and membrane, shows that the degree of inhibition depends mainly upon the concentration of the alcohol in the membrane. 1-butanol exerts a mixed-type inhibition; V_max decreases and K_m increases. Since also the K_m of the equilibrium transport increases upon the addition of the alcohol, the changes in the K_m of exchange transport cannot be attributed solely to the differently affected mobilities of the loaded and free carrier, but the affinity of glucose to the transport system is reduced. The transport system can bind two alcohol molecules. With one alcohol molecule bound the affinity of the transport system for the second alcohol molecule already increases. The nature of the bond of the alcohols to the transport system is discussed and possible explanations for the cooperative effect upon the binding of the second alcohol molecule are offered.     

Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer

The aspartate transporter from Pyrococcus horikoshii (Glt_Ph) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of Glt_Ph provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting Glt_Ph into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of Glt_Ph were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of Glt_Ph revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.     

Molecular aspects of the interaction between Mason—Pfizer monkey virus matrix protein and artificial phospholipid membrane

The Mason–Pfizer monkey virus is a type D retrovirus, which assembles its immature particles in the cytoplasm prior to their transport to the host cell membrane. The association with the membrane is mediated by the N‐terminally myristoylated matrix protein. To reveal the role of particular residues which are involved in the capsid‐membrane interaction, covalent labelling of arginine, lysine and tyrosine residues of the Mason–Pfizer monkey virus matrix protein bound to artificial liposomes containing 95% of phosphatidylcholine and 5% phosphatidylinositol‐(4,5)‐bisphosphate (PI(4,5)P₂) was performed. The experimental results were interpreted by multiscale molecular dynamics simulations. The application of these two complementary approaches helped us to reveal that matrix protein specifically recognizes the PI(4,5)P₂ molecule by the residues K20, K25, K27, K74, and Y28, while the residues K92 and K93 stabilizes the matrix protein orientation on the membrane by the interaction with another PI(4,5)P₂ molecule. Residues K33, K39, K54, Y66, Y67, and K87 appear to be involved in the matrix protein oligomerization. All arginine residues remained accessible during the interaction with liposomes which indicates that they neither contribute to the interaction with membrane nor are involved in protein oligomerization. Proteins 2016; 84:1717–1727. © 2016 Wiley Periodicals, Inc.     

Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C₁₀E₈), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/ phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX₁₀₀, C₁₂E₈ and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX₁₀₀-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when T₁₀₀ was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.     

A23187—Channel behaviour: Fluorescence study

Pyranine entrapped soylipid liposomes have been used as a model system to study the proton transport across membrane in the presence of A23187, a carboxylic ionophore specific for electroneutral exchange of divalent cations. An apparent rate constant (k_app) for transport of protons has been determined from the rate of change of fluorescence intensity of pyranine by stopped flow rapid kinetics in the presence of proton gradient The variation of k_app has been studied as a function of ionophore concentration and the results have been compared with gramicidin—a well known channel former under the similar experimental conditions. The rates thus obtained showed that A23187 is not only a simple carrier but also shows channel behaviour at high concentration of ionophore.     

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.     

Stability of diether C(25,25) liposomes from the hyperthermophilic archaeon Aeropyrum pernix K1

Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C_25,25 liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85 °C to 98 °C, only 10% of entrapped calcein was released. After 10 h at 90 °C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60 °C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C_25,25 liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C_25,25 liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65 °C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C_25,25 liposomes reveal a very broad and endothermic transition in the temperature range from 0 °C to 40 °C.