Tethered Bilayer Lipid Research Articles

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.

Tethered Lipid Bilayers within Porous Si Nanostructures: A Platform for (Optical) Real-Time Monitoring of Membrane-Associated Processes

The importance of cell membranes in biological systems has prompted the development of artificial lipid bilayers, which can mimic the cellular membrane structure. Supported lipid bilayers (SLBs) have emerged as a promising avenue for studying basic membrane processes and for possible biotechnological applications. Conventional methods for SLB formation involve the spreading of lipid vesicles on hydrophilic solid supports. Herein, a facile approach for the construction of tethered SLB within an oxidized porous Si (pSiO2) nanostructure, avoiding liposome preparation, is presented. We employ a two-step lipid self-assembly process, in which a first lipid layer is tethered to the pore walls resulting in a highly stable monolayer. A subsequent solvent exchange step induces the self-assembly of the unbound lipids into a robust SLB. Formation of pSiO2-SLB is confirmed by fluorescence resonance energy transfer (FRET), and the properties of the confined SLB are characterized by environment-sensitive fluorophores. The unique optical properties of the pSiO2 support are employed to monitor in real time the partitioning of a model amphiphilic molecule within the SLB via reflective interferometric Fourier transform spectroscopy (RIFTS) method. These self-reporting SLB platforms provide a highly generic approach for bottom-up construction of complex lipid architectures for performing biological assays at the micro- and nanoscale.

Nanoscale ion sequestration to determine the polarity selectivity of ion conductance in carriers and channels.

The nanoscale spacing between a tethered lipid bilayer membrane (tBLM) and its supporting gold electrode can be utilized to determine the polarity selectivity of the conduction of ion channels and ion carriers embedded in a membrane. The technique relies upon a bias voltage sequestering or eliminating ions, of a particular polarity, into or out of the aqueous electrolyte region between the gold electrode and the tethered membrane. A demonstration is given, using ac swept frequency impedance spectrometry, of the bias polarity dependence of the ionophore conductance of gramicidin A, a cationic selective channel, and valinomycin, a potassium ion selective carrier. We further use pulsed amperometry to show that the intrinsic voltage dependence of the ion conduction is actually selective of the polarity of the transported ion and not simply of the direction of the ionic current flow.

Peptide-induced formation of a tethered lipid bilayer membrane on mesoporous silica

Tethered bilayer lipid membranes (tBLMs) on solid supports have substantial advantages as models of artificial cell membranes for such biomedical applications as drug delivery and biosensing. Compared with untethered lipid membranes, tBLMs have more space between substrate and the bilayer and greater stability. The purpose of this work was to use these properties to fabricate and characterize a zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid tBLM containing 2 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-maleimide(poly(ethylene glycol))-2000 (DSPE-PEG2000-NHS) lipid tethers on a 3-aminopropyltrimethoxysilane-modified mesoporous silica substrate. A quartz crystal microbalance with dissipation monitoring was used to monitor the process of vesicle adsorption and tBLM self-assembly, and atomic force microscopy was performed to characterize the structural properties of the tBLM obtained. Whereas tether-containing lipid vesicles ruptured neither spontaneously nor as a result of osmotic shock, introduction of an amphipathic α-helical (AH) peptide induced vesicle rupture and subsequent tBLM formation. Taken together, our findings suggest that the AH peptide is an efficient means of rupturing vesicles of both simple and complex composition, and is, therefore, useful for formation of tBLMs on solid and mesoporous materials for applications in biotechnology.

Size dependent disruption of tethered lipid bilayers by functionalized polystyrene nanoparticles

Molecular interactions between engineered nanomaterials (ENM) and biomembranes are not well understood. This study investigated the effects of particle size and surface functional group on polystyrene nanoparticles' (PNPs) potency for biomembrane disruption. Electrochemical impedance spectroscopy was used to measure changes in the electrical resistance (Rm) of a tethered bilayer lipid membrane BLM (tBLM) composed of 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC) following PNP exposure. All PNPs tested triggered a decline in the Rm that could be described using an exponential-decay model. Statistical hierarchical clustering analysis of two model parameters (exponential rate constant and fractional loss of Rm) could distinguish between the PNPs based on both size and surface functional group. For COOH modified nanoparticles, 20nm PNPs were more potent in reducing Rm than 100nm PNP. However, for amidine modified nanoparticles, 120nm PNPs were more potent in reducing Rm than 23nm PNP. The COOH modified PNPs were more potent in reducing Rm than amidine modified PNP, which tended to aggregate following exposure to a tBLM. Ultra performance liquid chromatography–mass spectroscopy analysis suggested that the aggregation may have been triggered by DOPC that was removed from the tBLM by the amidine PNP.

The assembly and use of tethered bilayer lipid membranes (tBLMs).

Because they are firmly held in place, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cornell et al. Nature 387(6633): 580-583, 1997). Here we describe the procedures required to assemble and test tethered lipid bilayers that can incorporate various lipid species, peptides, and ion channel proteins.

Evaluation of Young’s Modulus of Tethered 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Membranes Using Atomic Force Spectroscopy

Unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with varying 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio) propionate] (DSPE-PEG-PDP) concentration between 0 mol % and 24 mol % were assembled on atomically flat template-stripped gold (TS Au) surfaces. Force spectroscopy, using an atomic force microscope (AFM), of the resulting tethered lipid bilayer membranes (tLBMs) in buffer provided information regarding mechanical response as a function of tethering molecule, DSPE-PEG-PDP, concentration. Young’s modulus was determined by fitting the force–indentation curve with a recently modified Sneddon model that corrects for contributions from the substrate underneath. At low concentrations, Young’s modulus is lower than that of a supported POPC LBM, i.e., directly sitting on a solid substrate. The decrease in modulus is attributed to increased membrane fluidity as coupling between the tLBM and solid substrate is reduced by the incorporation of DSPE-PEG-PDP tethering groups. From the determined Young’s modulus values, the PEG chain conformation is found to dominate tLBM rigidity at concentrations above 6 mol %. Analysis of AFM force spectroscopy data indicates that the poly(ethylene glycol) (PEG) mushroom to brush transition occurs near 6 mol %, and this leads to first softening and then abrupt stiffening of tLBMs at higher DSPE-PEG-PDP concentration associated with the transition. When DSPE-PEG-PDP concentration is increased to 24 mol %, AFM topography and Young’s modulus appear correlated with another phase transition; AFM topography images are consistent with a bilayer disk structure with DSPE-PEG-PDP segregated at the rim of the disk.

Characterization of the Electron Transport Chain Metabolon Bioelectrocatalysis

The electron transport chain is a group of membrane redox enzymes that are the driving force for the production of energy in mitochondria. Several theories and models have circulated different structures of the enzymes in the inner membrane.1 The three enzyme complexes form a supercomplex or respirasome, where Complex I, dimer Complex III, and various copies of Complex IV are electrostatically, hydrophobically and catalytically connected. While structural, kinetic, and genetic evidence prove the formation of the supercomplex, characterization of the bioelectrocatalysis has never been attempted of the three enzymes together on an electrode.2 The supercomplex contains multiple redox cofactors of the individual complex that span the lipid membrane and can come in contact with the electrode surface. By purifying the electron transport chain supercomplex, we have been able to reconstruct the inner membrane and immobilizing it onto a gold electrode. A polymer tethered lipid bilayer allows for the study of the natural bioelectrocatalysis of the large transmembrane proteins. With the addition of oxidized cytochrome c, Complex IV gives a catalytic response indicating that Complex III and Complex IV are active. With the addition of NADH and oxidized cytochrome c, the catalytic peaks increase. This indicates that all three enzymes are working together increasing their redox activities at the surface of the electrode. The supercomplex is further confirmed by inhibition of CIII and CIV, eliminating all activity of the enzymes at the electrode surface. This evidence shows how dependent the enzymes are on each other for efficient catalytic activity. The characterization of the supercomplex of these transmembrane redox enzymes and their bioelectrocatalysis on the electrode is the first for studying a natural metabolon electrochemically.

Preparation of Tethered-Lipid Bilayers on Gold Surfaces for the Incorporation of Integral Membrane Proteins Synthesized by Cell-Free Expression

There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cell-free expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor.

Voltage- and Calcium-Dependent Toxin Translocation Across a Tethered Lipid Bilayer

We report the design of novel biomimetic membrane model and its use to characterize in vitro the translocation process of bacterial toxin, the adenylate cyclase (CyaA) from Bordetella pertussis.The membrane was assembled over a calmodulin (CaM) layer and exhibits the fundamental characteristics of a biological membrane separating two cis and trans compartments. SPR was used to monitor the membrane interaction of the CyaA toxin, while the activation of the catalytic activity of CyaA by the tethered CaM was used as a probe of its translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent upon the presence of calcium, and upon application of a negative trans-membrane potential, in good agreement with prior studies done on eukaryotic cells. These results demonstrate that CyaA does not require any eukaryotic components to translocate across a membrane, and suggest that CyaA is electrophoretically transported across the bilayer by the transmembrane electrical field. To our knowledge, this work constitutes the first in vitro demonstration of protein translocation across a tethered lipid bilayer. This biomimetic assembly opens new opportunities to explore the molecular mechanisms of protein translocation across biological membranes.

Tethered Lipid Bilayer Gates: Toward Extended Retention of Hydrophilic Cargo in Porous Nanocarriers

A high‐performance molecular gating system for efficient capping and delivery of hydrophilic cargo is reported. It integrates a mesoporous silica nanoparticle core and a lipid bilayer (LB) shell by covalent tethering via a hyperbranched polyethylenimine (PEI) cushion. When using calcein as a general model for hydrophilic drug molecules, a high payload is loaded into the porous structure due to greatly enhanced concentration of amino groups on the pore walls. Surprisingly, LB non‐disruptively resides on the porous surface in this system, despite the strong positive charge from PEI, originating from the covalent tethering of the inner leaflet, as well as preferential spanning over the pore openings facilitated by the stretching of PEI chains on the particle surface. An unprecedented high retention of negatively charged hydrophilic guest molecules after up to 1 week is consequently achieved, even in the presence of a membrane disrupting agent. Furthermore, a PEI‐induced charge conversion at neutral pH is conferred to the particles using a zwitterionic PC lipid as the outer leaflet of LB. Interestingly, the corresponding nanocarriers are able to promote cargo escape from endosomes. Subsequent delivery of the loaded hydrophilic cargo to the cytoplasm is observed despite the tight retention under extracellular conditions.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.

Size Dependent Disruption of Tethered Lipid Bilayers by Carboxylate-Modified Polystyrene Nanoparticles

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DNA-Based Patterning of Tethered Membrane Patches

Solid-supported lipid bilayers are useful model systems for mimicking cellular membranes; however, the interaction of the bilayer with the surface can disrupt the function of integral membrane proteins. As a result, many groups have introduced tethered lipid bilayers, which retain the proximity to the surface, enabling surface-sensitive techniques, but physically distance the bilayer from the surface. We have recently developed a method for spatially separating a lipid bilayer from a solid support using DNA lipids (Chung and Boxer et al., J. Struct. Biol., 2009). In this system, a DNA strand is covalently attached to a silane-modified glass slide or SiO2 wafer. The complementary DNA strand conjugated to a lipid moiety is inserted into giant unilamellar vesicles (GUVs), and the DNA-modified GUVs hybridize to the strands on the surface, inducing flattening and rupture of the GUV to a planar tethered lipid bilayer. However, the location of the patch is random, determined by where the DNA-GUV initially binds with its complement. To allow greater versatility and control, we sought a way to pattern tethered membrane patches. We present a method for creating spatially distinct tethered membrane patches on a glass slide using microarray printing. Surface-reactive DNA sequences are spotted onto the slide, incubated to covalently link the DNA to the surface, and DNA-GUVs patches are formed selectively on the printed DNA. Different DNA sequences can be printed on the same slide, creating a unique handle on each GUV patch. This handle enables the creation of patches of different lipid compositions, dyes, and/or DNA-lipid sequences in adjacent but distinct areas, and the control over the placement of the tethered lipid bilayer potentially allows interfacing with devices. This approach would also enable rapid screening of different patches in protein binding assays and as targets for membrane fusion.

Coarse-grained molecular dynamics simulation of tethered lipid assemblies

Using coarse-grained molecular dynamic simulations based on the MARTINI force field, we study the self-assembly of free dipalmitoylphosphatidylcholine (DPPC) lipids onto PEGylated lipids tethered to a solid substrate. We show that upon increasing lipid concentration, structural transitions occur from tethered spherical nanoparticles, to tethered cylinders and bicelles, to a tethered lipid bilayer with pores, to an unporated tethered lipid bilayer, and finally to a tethered lipid bilayer with liposomes on top of it. The simulation results compare well with structures inferred from experimental observations. In addition, we demonstrate the structural stability and local fluidity of the tethered lipid bilayer.

Biotechnology Applications of Tethered Lipid Bilayer Membranes

The importance of cell membranes in biological systems has prompted the development of model membrane platforms that recapitulate fundamental aspects of membrane biology, especially the lipid bilayer environment. Tethered lipid bilayers represent one of the most promising classes of model membranes and are based on the immobilization of a planar lipid bilayer on a solid support that enables characterization by a wide range of surface-sensitive analytical techniques. Moreover, as the result of molecular engineering inspired by biology, tethered bilayers are increasingly able to mimic fundamental properties of natural cell membranes, including fluidity, electrical sealing and hosting transmembrane proteins. At the same time, new methods have been employed to improve the durability of tethered bilayers, with shelf-lives now reaching the order of weeks and months. Taken together, the capabilities of tethered lipid bilayers have opened the door to biotechnology applications in healthcare, environmental monitoring and energy storage. In this review, several examples of such applications are presented. Beyond the particulars of each example, the focus of this review is on the emerging design and characterization strategies that made these applications possible. By drawing connections between these strategies and promising research results, future opportunities for tethered lipid bilayers within the biotechnology field are discussed.

Membrane Tethering and Nucleotide-dependent Conformational Changes Drive Mitochondrial Genome Maintenance (Mgm1) Protein-mediated Membrane Fusion

Cellular membrane remodeling events such as mitochondrial dynamics, vesicle budding, and cell division rely on the large GTPases of the dynamin superfamily. Dynamins have long been characterized as fission molecules; however, how they mediate membrane fusion is largely unknown. Here we have characterized by cryo-electron microscopy and in vitro liposome fusion assays how the mitochondrial dynamin Mgm1 may mediate membrane fusion. Using cryo-EM, we first demonstrate that the Mgm1 complex is able to tether opposing membranes to a gap of ∼15 nm, the size of mitochondrial cristae folds. We further show that the Mgm1 oligomer undergoes a dramatic GTP-dependent conformational change suggesting that s-Mgm1 interactions could overcome repelling forces at fusion sites and that ultrastructural changes could promote the fusion of opposing membranes. Together our findings provide mechanistic details of the two known in vivo functions of Mgm1, membrane fusion and cristae maintenance, and more generally shed light onto how dynamins may function as fusion proteins.

Elucidating Driving Forces for Liposome Rupture: External Perturbations and Chemical Affinity

Atomic force microscopy (AFM) studies under aqueous buffer probed the role of chemical affinity between liposomes, consisting of large unilamellar vesicles, and substrate surfaces in driving vesicle rupture and tethered lipid bilayer membrane (tLBM) formation on Au surfaces. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio) propionate] (DSPE-PEG-PDP) was added to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles to promote interactions via Au-thiolate bond formation. Forces induced by an AFM tip leading to vesicle rupture on Au were quantified as a function of DSPE-PEG-PDP composition with and without osmotic pressure. The critical forces needed to initiate rupture of vesicles with 2.5, 5, and 10 mol % DSPE-PEG-PDP are approximately 1.1, 0.8, and 0.5 nN, respectively. The critical force needed for tLBM formation decreases from 1.1 nN (without osmotic pressure) to 0.6 nN (with an osmotic pressure due to 5 mM of CaCl(2)) for vesicles having 2.5 mol % DSPE-PEG-PDP. Forces as high as 5 nN did not lead to LBM formation from pure POPC vesicles on Au. DSPE-PEG-PDP appears to be important to anchor and deform vesicles on Au surfaces. This study demonstrates how functional lipids can be used to tune vesicle-surface interactions and elucidates the role of vesicle-substrate interactions in vesicle rupture.

Electron Transport in Supported and Tethered Lipid Bilayers Modified with Bioelectroactive Molecules

Tethered bilayer lipid membranes (tBLMs) are commonly used as model membranes. However, in biophysical studies, free-standing membranes (“black” lipid membranes) are more realistic models of cellular processes. In this article, we discuss the rates of electron transfer in both types of bilayer lipid membranes. These BLMs were then modified using two very important mitochondrial membrane-associated molecules: ubiquinone-10 (UQ10) and α-tocopherol (VitE). The electron transfer rates in the unmodified films were studied with three redox couples, Fe(CN)6(3-/4-), Ru(NH3)6(3+/2+), and NAD+/NADH, using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The rate of electron transfer in the modified films was studied using the biologically relevant NAD+/NADH electroactive couple using the same methods. It is shown that when the BLMs are modified with only UQ10, it is possible to observe electron transfer. However, when the antioxidant VitE is added to the modification, the electron transfer provided by UQ10 is inhibited. Following initial studies using CV, a comparison of electron transfer theory and data was used to investigate this phenomenon in more detail, using EIS data. The standard rate constant caused by electron tunneling across the film, k(th)(0), depends on the value of β used. Two different values of the potential independent electron tunneling coefficient, β, were fitted, and it is shown that a β value half of those usually reported in literature (refereed here as β(app)) gives better agreement between the theory and the experimental results. The unmodified films present k(th)(0) values on the order of 10(-15) cm s(-1) when β = 0.72 Å(-1) and k(th)(0) values on the order of 10(-9) cm s(-1) when β(app) = 0.38 Å(-1). For the modified films, the values of k(th)(0) are on the order of 10(-15) cm s(-1) when β = 0.72 Å(-1) and 10(-9) cm s(-1) for β(app) = 0.38 Å(-1). The experimental electron transfer rate constant, k(app)(0), is on the order of 10(-8) cm s(-1) for unmodified and modified (with (i) UQ10, (ii) VitE, and (iii) UQ10 + VitE) films.

A molecular machine biosensor: Construction, predictive models and experimental studies

This paper describes the construction, operation and predictive modeling of a molecular machine, functioning as a high sensitivity biosensor. Embedded gramicidin A (gA) ionchannels in a self-assembled tethered lipid bilayer act as biological switches in response to target molecules and provide a signal amplification mechanism that results in high sensitivity molecular detection. The biosensor can be used as a rapid and sensitive point of care diagnostic device in different media such as human serum, plasma and whole blood without the need for pre and post processing steps required in an enzyme-linked immunosorbent assay. The electrical reader of the device provides the added advantage of objective measurement. Novel ideas in the construction of the molecular machine, including fabrication of biochip arrays, and experimental studies of its ability to detect analyte molecules over a wide range of concentrations are presented. Remarkably, despite the complexity of the device, it is shown that the response can be predicted by modeling the analyte fluid flow and surface chemical reactions. The derived predictive models for the sensing dynamics also facilitate determining important variables in the design of a molecular machine such as the ion channel lifetime and diffusion dynamics within the bilayer lipid membrane as well as the bio-molecular interaction rate constants.