Large Unilamellar Lipid Vesicles Research Articles

Coupling of Leaflet Structure in Asymmetric Lipid Vesicles

Lipid asymmetry is a hallmark of biological membranes. In particular, prototypical mammalian plasma membranes are known to be composed of an outer leaflet enriched in cholinephospholipids, while the majority of the aminophospholipids are confined to the inner leaflet. Asymmetric large unilamellar lipid vesicles (aLUVs), produced via cyclodextrin-mediated lipid exchange, are a new platform for more realistic mimics of biological membranes. These systems were shown to be stable over several days and have already been investigated by elastic scattering techniques (small-angle neutron and X-ray scattering; SANS/SAXS), providing insight into structural properties of the individual leaflets. One of the enduring questions concerning plasma membrane architecture and lipid asymmetry is the possibility of bilayer leaflets being coupled to each other, which may influence a number of physiological processes that require communication between interior and exterior of the cell. However, in the physiologically relevant fluid phase no evidence of structural coupling has yet been reported from scattering studies. In this work, we explore the role of hydrocarbon chain interdigitation as a potential trigger for transleaflet coupling. We use combinations of dipalmitoylphosphatidylcholine (DPPC) in the inner leaflet and mixed lipids with varying chain length mismatch in the outer leaflet, in particular C16:0/C18:1 PC (POPC), C18:0/C18:1 PC (SOPC), C18:0/C14:0 (SMPC), C14:0/C18:0 (MSPC) and C16:0/C14:0 PC (PMPC). This entails different interdigitation states of the mixed-chain lipids into the inner leaflet. We present consequences on transbilayer coupling as observed from leaflet specific structural data and thermotropic behavior of these systems.

Intriguing interaction of a cyclometalated dinuclear Ir(III) complex bridged by imidazolyl phenanthroline with anionic and cationic lipid vesicles

Interaction of a cyclometalated dinuclear Ir(III) complex with large and small unilamellar lipid vesicles (LUVs and SUVs) was studied in aqueous medium. This class of Ir(III) coordination complexes have potential biological implications and are frequently used as therapeutic agents and biomarkers. The complex emits due to triplet metal-to-ligand charge transfer (3MLCT) (dπIr→π*imidazolylphenanthroline). The weak emission of the complex, in aqueous medium, increases profusely on interaction with anionic lipid vesicles. Luminescence, however, gets quenched on application of cationic vesicles. The present study shows that charged lipid vesicles interact differently with the dinuclear Ir(III) complex and, in turn, affect its 3MLCT emission. The outcome of the present experiment spectroscopically describes the changes in photophysical aspects of the Ir(III) complex due to the application of the lipid vesicles in aqueous environment.

The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate

We have investigated the effect of lipid composition on interactions between cytochrome bo3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers.

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial, which are crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to significant insights into vital membrane phenomena such as domain formation, passive permeation, and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatial and temporal. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular understanding of lipid membranes. In particular, the synergy between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. Here, we describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidities. Specifically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for studies of more complex membrane systems.

Applying Fluorescence Correlation Spectroscopy to Investigate Peptide-Induced Membrane Disruption.

There is considerable interest in understanding the interactions of antimicrobial peptides with phospholipid membranes. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that can be used to gain insight into these interactions. Specifically, FCS can be used to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain information about whether leakage occurs due to localized membrane perturbations or global membrane destabilization. Here, we outline a detailed step-by-step protocol on how to optimally implement an FCS-based leakage assay. To make the protocol easily accessible to other researchers, it has been supplemented with a number of practical tips and tricks.

Size fractionation and size characterization of nanoemulsions of lipid droplets and large unilamellar lipid vesicles by asymmetric-flow field-flow fractionation/multi-angle light scattering and dynamic light scattering

Asymmetric-flow field-flow fractionation technique coupled to a multi-angle light-scattering detector (AF4-MALS) was used together with dynamic light-scattering (DLS) in batch mode and transmission electron microscopy (TEM) to study the size characteristics of the trioleoylglycerol lipid droplets covered by a monolayer of sphingomyelin and cholesterol, in water phase. These lipid droplet nanoemulsions (LD) were formed by ultrasonication. In parallel, the size characteristics of large unilamellar lipid vesicles (LUV) prepared by extrusion and composed of sphingomyelin and cholesterol were determined. LD and LUV were prepared at two different molar ratios (1/1, 4/1) of sphingomyelin and cholesterol. In AF4-MALS, various cross-flow conditions and mobile phase compositions were tested to optimize the separation of LD or LUV particles. The particle radii, R, as well as the root-mean-square radii, Rrms, of LD and LUV were determined by AF4-MALS, whereas the hydrodynamic radii, Rh, were obtained by DLS. TEM visualization revealed round shape particles of LD and LUV.

The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions.

Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 with Thermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid. The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.

Single-vesicle detection and analysis of peptide-induced membrane permeabilization.

The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin 2. The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied. These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying peptide-induced leakage from lipid vesicles.

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.

Shear-induced membrane fusion in viscous solutions.

Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We present evidence that shear forces in a viscous solution can induce lipid bilayer fusion. The fusion of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes is observed in Couette flow with shear rates above 3000 s(-1) provided that the medium is viscous enough. Liposome samples, prepared at different viscosities using a 0-50 wt % range of sucrose concentration, were studied by dynamic light scattering, lipid fusion assays using Förster resonance energy transfer (FRET), and linear dichroism (LD) spectroscopy. Liposomes in solutions with 40 wt % (or more) sucrose showed lipid fusion under shear forces. These results support the hypothesis that under suitable conditions lipid membranes may fuse in response to mechanical-force-induced stress.

Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte

In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant ‘on’ and ‘off’ binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new ‘hybrid’ delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles.Vesicles are artificially prepared, small lipid membrane-enclosed sacks that can store substances. As vesicles encapsulate an aqueous solution inside a partly hydrophobic membrane, they can carry both hydrophobic and hydrophilic molecules. Due to their unique properties, liposomes are often used as model systems to create or study artificial cells, as well as drug delivery vehicles. In order to transport nucleic acids, proteins or other cargo, the vesicles should exhibit mobility. In the present paper we propose a new delivery strategy, using the motion of spermatozoa to transport attached vesicles into the oocyte during fertilization. To use spermatozoa as a vesicle-transport system, we have to accomplish three goals. One, the lipid composition of the vesicle membranes needs to be created such that they can bind to the sperm cells. Two, the spermatozoa should still be able to swim with the extra loaded vesicle cargo. And three, the bound vesicles should not inhibit the capability of sperm cells to fertilize. In this study, vesicles started to show binding after 20mol% neutral lipids were substituted with either positive, negative, cerebroside or ganglioside lipids. A new ‘hybrid’ delivery system has been created in which vesicles are bound to spermatozoa in physiological conditions with no significant alterations in sperm motility and function. Moreover, with IVF experiments in the mouse model, it was confirmed that spermatozoa remained capable of fertilizing while delivering vesicles simultaneously.

Phosphatidylinositol 4,5-Bisphosphate Decreases the Concentration of Ca2+, Phosphatidylserine and Diacylglycerol Required for Protein Kinase C α to Reach Maximum Activity

The C2 domain of PKCα possesses two different binding sites, one for Ca2+ and phosphatidylserine and a second one that binds PIP2 with very high affinity. The enzymatic activity of PKCα was studied by activating it with large unilamellar lipid vesicles, varying the concentration of Ca2+ and the contents of dioleylglycerol (DOG), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphadidylserine (POPS) in these model membranes. The results showed that PIP2 increased the Vmax of PKCα and, when the PIP2 concentration was 5 mol% of the total lipid in the membrane, the addition of 2 mol% of DOG did not increase the activity. In addition PIP2 decreases K0.5 of Ca2+ more than 3-fold, that of DOG almost 5-fold and that of POPS by a half. The K0.5 values of PIP2 amounted to only 0.11 µM in the presence of DOG and 0.39 in its absence, which is within the expected physiological range for the inner monolayer of a mammalian plasma membrane. As a consequence, PKCα may be expected to operate near its maximum capacity even in the absence of a cell signal producing diacylglycerol. Nevertheless, we have shown that the presence of DOG may also help, since the K0.5 for PIP2 notably decreases in its presence. Taken together, these results underline the great importance of PIP2 in the activation of PKCα and demonstrate that in its presence, the most important cell signal for triggering the activity of this enzyme is the increase in the concentration of cytoplasmic Ca2+.

Measuring peptide translocation into large unilamellar vesicles.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

Comparison of Various Methods for Determining Functional Activity of the Diphtheria Toxin T-Domain

The function of diphtheria toxin translocation (T) domain is a transfer of catalytic domain across endosomal membrane in response to acidification. The goal of this study is to develop a simple and reliable in vitro functional assay for the T-domain, one that can be used to investigate structure-function relationships using interactions of various mutants with lipid bilayers of various compositions. Traditionally the activity of the T-domain is estimated either by measuring conductance in planar lipid bilayers or by measuring the release of fluorescent markers from large unilamellar lipid vesicles. We have applied these measurements to a series of T-domain mutants with substitutions in His residues involved in modulating pH-dependent refolding and insertion of the protein. The comparison of the results with those obtained in cell cytotoxicity assay performed with full length toxin revealed lack of correlation of the leakage-based assay with other assays. Our data suggest that release of fluorescent markers is related to the stress induced by the interfacial binding, rather than by the insertion of the protein in a functional conformation. To resolve the contradiction between cellular and in vitro measurements we have developed a translocation test based on the cleavage of the N-terminal part of T-domain upon its translocation into thrombin-loaded vesicles. Application of this test to a series of mutants correlated well with results of cytotoxicity. In the other set of experiments we have investigated lipid dependence - non-linear changes with surface potential (which is in contrast with leakage measurements) confirming our previous suggestion that formation of the final inserted state is modulated by anionic lipids. NIH GM069783 (ASL), GM-29210 (AF), AI-022021(RGC) and Fulbright Foundation.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: A comparative study of spinach SoPIP2;1 and E. coli AqpZ

This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.

Possible Membrane-Segragation of PPI/PC Membranes Induced by Calcium Ions

Abstract Calcium-Dependent possible membrane segregation in PPI/PC membranes was studied by using NBD-labeled various phosphoinositides (PPIs). The NBD fluoropore is known that its fluorescence emission signal is reduced as the fluorophore concentration increases. Using this effect, we try to interpret the NBD-incorporated PPIs accumulation in the PC membrane by calcium ions. Either small or large unilamellar lipid vesicles composed of phosphatidylcholine (PC) and NBD-incorporated various phospoinositides (PI, PIP or PIP2) at different molar ratios were suspended in 0.1 M NaCl/10 mM Hepes at pH 7.4. Then, different concentrations of calcium chloride were made in each vesicle suspension and the NBD emission signals of these phospholipid vesicles were measured by use of a fluorimeter in time at room temperature. As a reference experiment, the vesicle suspension containing 40 µM EDTA without additi of Ca2+ was also measured. The molar concentrations of PPIs in PC membrane were 4% and 8% (mole ratio of PPI to PC). The NBD emission signal decreases took rather long time. The higher molar ratio of PPI in PC gave shorter time course. The larger signal decreases were observed at higher concentrations of Ca2+ and the vesicle suspension containing EDTA gave more or less a constant and the highest emission signal. The reduction of NBD emission signals was also dependent on the different types of PIPs used. The PIP2 containing PC membrane showed the largest changes in the fluorescence signal decreases. The order was PIP2 > PIP > PI. Form these results, we interpret that PPIs in PC membranes are segragated by themselves with Ca2+.

Using giant unilamellar lipid vesicle micro-patterns as ultrasmall reaction containers to observe reversible ATP synthesis/hydrolysis of F 0F 1-ATPase directly

F 0F 1-ATPase within chromatophores, which was labeled with pH-sensitive quantum dots, was encapsulated in large unilamellar lipid vesicles (LUVs) through reverse-phase evaporation. Then a microarray of chromatophore-containing LUVs was created using a micro-contact printing (μ-CP) technique. Through controlled dehydration–rehydration of the lipid patterns, a microarray of single chromatophore-containing giant unilamellar lipid vesicles (GUVs) was formed with desired size and uniform shape. The reversible ATP synthesis/hydrolysis of F 0F 1-ATPase in GUVs was directly observed by fluorescence microscopy through the fluorescence intensity increase/decrease in the pH-sensitive quantum dots labeled on the outer surface of the chromatophore. To the best of our knowledge, this is the first direct observation of the reversible behavior of F 0F 1-ATPase at the bulk scale.

Is Lipid Bilayer Binding a Common Property of Inhibitor Cysteine Knot Ion-Channel Blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (Δ G) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of Δ G dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep Δ G dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed Δ G shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.

Properties of Mixed Lipid Monolayers Assembled on Hydrophobic Surfaces through Vesicle Adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.