A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes (original) (raw)
Related papers
Journal of visualized experiments : JoVE, 2018
Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to ...
Charge-selective membrane protein patterning with proteoliposomes
RSC Adv., 2014
A novel method to fabricate transmembrane protein (TP) embedded lipid bilayers using microcontact printing and applying proteoliposomes to different types of substrates, has been developed. The electrostatic interaction between the negatively charged proteoliposome and the substrate, which had been positively functionalized by the microcontact printing, allowed the formation of TP-embedded, patterned lipid bilayers. The positively charged amino functional group on the substrate did effectively attract the negatively charged vesicles, inducing them to be adsorbed and subsequently ruptured to form a giant mosaic lipid bilayer, resulting in an immobilized TP-embedded lipid layer precisely on the targeted patterns, which were backfilled with zwitterionic lipid bilayer. The rapid and highly selective recognition of the charged liposomes was visualized, and the biological functions from the TPs in the lipid matrix were also observed.
Biochemical and Biophysical Research Communications, 2011
Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10 5 FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm 2 within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.
Inherently Tunable Electrostatic Assembly of Membrane Proteins
Nano Letters, 2008
Membrane proteins are a class of nanoscopic entities that control the matter, energy, and information transport across cellular boundaries. Electrostatic interactions are shown to direct the rapid co-assembly of proteorhodopsin (PR) and lipids into long-range crystalline arrays. The roles of inherent charge variations on lipid membranes and PR variants with different compositions are examined by tuning recombinant PR variants with different extramembrane domain sizes and charged amino acid substitutions, lipid membrane compositions, and lipid-to-PR stoichiometric ratios. Rational control of this predominantly electrostatic assembly for PR crystallization is demonstrated, and the same principles should be applicable to the assembly and crystallization of other integral membrane proteins.
Biochimica Et Biophysica Acta-biomembranes
Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K+ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~ 80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of 15N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.► Membrane mimicking additives promote cell-free synthesis of soluble membrane proteins. ► Lipid–protein nanodiscs were compared with detergent micelles, bicelles and liposomes. ► Nanodiscs facilitate the correct folding of synthesized membrane proteins. ► Partial disintegration of nanodiscs composed of unsaturated lipids was observed. ► NMR studies of proteins co-translationally incorporated into nanodiscs are possible.
Journal of the American Chemical Society, 2009
Nanomedicine utilizes nanoscale materials and principles to effect medical intervention at the molecular scale with the goal of curing diseases or repairing tissues. Drug delivery is arguably one of the most important and promising areas in nanomedicine. 1 Among the many recently developed drug carriers, inorganic nanomaterials, 2,3 in particular amorphous mesoporous silica nanoparticles, are very attractive because of their biocompatibility combined with high surface area and pore volume as well as uniform, tunable pore diameters and surface chemistries. 4 Through simple electrostatic interactions, drugs can be loaded at very high concentrations. Recently, such porous silica particles have been utilized to deliver a wide range of drugs and therapeutic agents, including chemotherapy drugs, proteins, and DNA. [5] Achieving high drug loading, however, is only one facet of the drug delivery problem. It is equally important that loaded drugs are retained and protected before reaching target tissues or cells to maximize drug efficacy and minimize toxicity. For drugs loaded by electrostatic interactions or physical adsorption in freely accessible pores, metabolites/ ions in the body fluid can displace the drugs ( , pathway 1), resulting in premature drug release. To avoid this, molecular-gating strategies based on coumarin, 8 azobenzenes, 9,10 rotaxanes, 11 polymers, 12, 13 and nanoparticles have been designed, wherein drugs are released only upon gate opening or removal.
Forming giant vesicles with controlled membrane composition, asymmetry, and contents
Proceedings of the National Academy of Sciences, 2011
Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.
High-level cell-free production of membrane proteins with nanodiscs
2014
Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the cotranslational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.
A Self-assembly Route for Double Bilayer Lipid Membrane Formation
ChemPhysChem, 2010
Solid supported lipid membranes provide a simple biomimetic model system that is suitable for studying a wide range of membrane related phenomena and importantly permits the use of a number of surface analytical techniques from AFM to impedance spectroscopy to be used in characterising such processes. Additionally, it is also believed that such systems could find application for drug screening, biosensing or in protein separation/crystallisation. [9] In nature there are however a number of situations in which double bilayers naturally occur, for example in mitochondria or complexes which span two lipid bilayers at gap junctions, [11] and for this reason it would be desirable to be able to create double bilayer mimics as an extension of the supported bilayer field. It has been previously demonstrated that one can create such structures using the Langmuir-Blodgett technique, [12] however, this has the drawback that it is not compatible with the incorporation of transmembrane proteins and the film is created by transfer through the air-water interface. Hence approaches based on self-assembly from vesicles would provide a significant advance in the type of applications that double bilayers could be used for. Murray et al. have recently demonstrated, a previously observed phenomenon, that a second bilayer can be assembled on top of a streptavidin protein film, attached to a first bilayer. Further, Chung et al. have also shown that giant unilamellar vesicle (GUV) rupture can lead to the formation of a second bilayer tethered to a first bilayer using complementary DNA sequences. Similarly, GUV rupture onto a lipid bilayer to form model intermembrane junctions has been reported by Kaizuka and Groves; and Tabaei et al. have demonstrated a method whereby DNA duplexes were used to tether multiple diskshaped lipid "bicelles" to a bilayer. While these systems could not be used for studying double bilayer phenomena directly, they demonstrate that the principle of achieving double bilayers via self-assembly is feasible. Here we describe a new method to form double bilayer lipid membranes (dBLMs) on solid supports using NHS/EDC chemistry [hydroxy-2,5-dioxopyrrolidine-3-sulfonicacid sodium salt (NHS) and N-Ethyl-N'-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) ]. In this approach, two biomembranes, one of which contains the amine-functionalised cholesterol derivative 1 and the other which contains the CO 2 H-funtionalised cholesterol derivative 2 are covalently joined together by an NHS/EDC mediated reaction as shown schematically in . Details of the synthesis and characterization of the cholesterol derivatives 1 and 2 are given in the Supporting Information. These dBLMs can be formed both on functionalized Au and on silicon oxide surfaces. Here we describe their formation on a silicon oxide surfaces, monitored using a combination of fluorescence microscopy and atomic force spectroscopy. Vesicles containing Egg PC, reagent 2, DOTAP, and the red fluorescently labeled lipid TR DHPE (molar ratio 63.5🔞18:0.5) were used to form a single supported bilayer on a glass cover slip in the usual way. As shown in a, the fluorescence of the TR DHPE within this first bilayer is clearly seen, using a Texas red filter, and is uniform, whilst an image of the same region using a FITC filter does not show any fluorescence . The lateral diffusion coefficient of the TR DHPE was obtained from fluorescence recovery after photobleaching (FRAP). The average value of D from three different samples was 1.3 AE 0.2 mm 2 s À1 with the mobile fraction of 93 %. The COOH groups of the reagent 2 within this first sBLM were activated by incubating for 15 min with NHS/EDC (5:1 ratio) solution. After thoroughly rinsing with Milli-Q water to remove the excess NHS/EDC, vesicles containing Egg PC, reagent 1 and green fluorescent lipid D291(molar ratio 71🔞1) were added. A reaction occurred between reagents 1 and 2 and a second bilayer was formed, covalently linked through amide bonds to the first bilayer. After rinsing, the double bilayer was investigated by fluorescence microscopy. Figure 2 c, taken with the Texas red filter, indicates the fluorescence of the inner bilayer, while Figure 2 d, taken with the FITC filter, shows the fluorescence of the outer bilayer.
The Journal of general physiology, 2016
Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo-electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in...