Incorporation Of Integral Membrane Proteins Research Articles

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions.

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane–membrane interactions and possible future research directions.

A Triple Role for a Bilayer: Using Nanoliposomes to Cross and Protect Cellular Membranes.

Thanks in large part to the seminal work of Steve White and his colleagues, we appreciate the "ordered complexity" of the lipid bilayer and how it impacts the incorporation of integral membrane proteins as well as more peripherally associated proteins. Steve's work also provides a vital foundation to tackle another challenge: cytotoxic oligomeric complexes which accumulate in various neurodegenerative diseases. These oligomers have a relatively fluid structure and interact with many different proteins in the cell, but their main target is thought to be the phospholipid membrane, either the plasma membrane or internal organelles such as the mitochondria. This fascinating encounter between two essentially fluid phases generates a more disordered membrane, and presumably promotes uncontrolled transport of small metal ions across the membrane barrier. Happily, this unwanted interaction may be suppressed by mobilizing the phospholipid bilayer into its own defense. Extruded nanolipoparticles (NLPs) consisting of DPPC lipids, cholesterol and PEG2000 are excellent vehicles to take up small "oligomer-bashing" hydrophobic molecules such as baicalein and transport them with increased half-life in the plasma and with markedly more efficient crossing of the blood-brain barrier. Thus the bilayer has a triple role in this account: a safe space for a reactive hydrophobic small molecule, a barrier to cross to deliver a drug payload and a target to protect against oligomer attacks. NLPs containing small hydrophobic molecules show great promise in combating neurodegenerative diseases in animal models and may serve as an example of the White approach: applying robust physical-chemical principles to deal with biological problems involving phospholipid membranes.

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.

Incorporation of Integral Membrane Proteins in Giant Lipid Vesicles by Swelling from a Protein-Loaded Hydrogel

While giant unilamellar lipid vesicles (GUVs) have proven to be useful tools for probing the properties of biomembranes, the incorporation of integral membrane proteins into GUVs of controlled composition has remained challenging. We have developed a simple method for incorporating integral membrane proteins into the GUV membrane. Detergent-solubilized proteins are dissolved in an agarose hydrogel, which is spread as a thin film on a solid substrate. Lipids of the desired membrane composition are then deposited on top of the partially dehydrated, protein-containing hydrogel. The entire system is swollen with buffer following previously published protocols for forming GUVs from hydrogel-supported lipid films. The final detergent concentration in the rehydrated system is significantly lower than the critical micellar concentration of the detergent used to solubilize the membrane protein, which is transferred to the lipid bilayer of the GUV. GUVs form reliably and with good yield at a wide range of buffer ionic strengths and lipid compositions. High-molecular weight species can be encapsulated within the GUV interiors by including them in the rehydration buffer. We have used this system to demonstrate the cholesterol-dependent phase segregation of the alpha-helical transmembrane proteins aquaporin SoPIP2;1, aquaporin-Z, and bacteriorhodopsin. In lipid systems expected to undergo liquid ordered-liquid disordered phase segregation, these three proteins all consistently co-segregate with the cholesterol-poor phase as cholesterol concentration is varied from 20 to 50 mol%. We have also incorporated the human glucose transporter GLUT1 into GUV membranes and demonstrated its function. GUVs were fabricated encapsulating an enzymatic glucose assay with a fluorescent output. These GUVs were then immobilized in a microfluidic channel and a buffer containing glucose was introduced. Fluorescence was observed only in those GUVs fabricated with GLUT1 present in the agarose substrate.

Structure and properties of tethered bilayer lipid membranes with unsaturated anchor molecules.

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection-absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative.

Supported Lipid Bilayers on Biocompatible Polysaccharide Multilayers

Solid-supported phospholipid bilayers are widely used as models to probe the function of cellular membranes and as platforms for membrane protein biosensing applications. Although supported lipid bilayers have a number of advantages in terms of ease of fabrication and characterization, the proximity of the membrane to the solid support is a limitation for applications that involve incorporation of integral membrane proteins. This limitation can be overcome by formation of supported lipid bilayers on soft polymer cushions. We have prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid (HA), on glass substrates. (CHI/HA)5 films were characterized by atomic force microscopy, giving an average thickness of 57 and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and pH 6.5 on (CHI/HA)5 films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was due to the formation of heterogeneous membranes with defects and pinned lipids, rather than to a layer of surface adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The formation of bilayers containing reconstituted integral membrane protein was compared on PEM and PEG films. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.

Solution NMR Spectroscopy and Protein Interaction Studies of Membrane Proteins in Nanodiscs

Insolubility of integral membrane proteins (IMPs) in buffer often prevents their investigation with biophysical methods developed for soluble proteins. First, solution NMR of large proteins or complexes is limited by slow rotational diffusion. IMPs in cellular membranes or liposomes are beyond the size limit of solution NMR. Therefore, detergent micelles or bicelles are often used for solubilization and NMR studies of IMPs. Unfortunately, detergents may destabilize the native structure or compromise the activity of proteins. Nanodiscs are a lipid-based, detergent free, relatively small and very promising new model membrane system. We inserted a recombinantly produced and 15N, 13C-labeled fragment of human CD4 into nanodiscs. This polypeptide comprises the membrane-spanning and the cytoplasmic domains of CD4. Our NMR data demonstrate the feasibility of solution NMR spectroscopy on IMPs in nanodiscs. Second, surface plasmon resonance (SPR) is predominantly utilized in interaction studies of soluble proteins. Incorporation of IMPs into nanodiscs provides a close to native environment to the membrane protein and results in a water-soluble proteolipid particle that might be amenable to standard SPR-based methodology. We reconstituted a decahistidine-tagged IMP into nanodiscs and studied binding between the nanodisc-inserted IMP and a PentaHis monoclonal antibody (mAb) immobilized on the surface of a CM5-sensorchip. For comparison, we also determined the affinity of the decahistidine-tagged soluble domain of the same IMP toward the immobilized PentaHis mAb. Binding affinities were almost identical in both cases. However, the association and dissociation rate constants were found to differ, which is in agreement with the distinct diffusion coefficients of the soluble analyte particles. Our data indicate that nanodisc-inserted IMPs can serve as analyte in interaction studies of membrane proteins.

In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS)

Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

Steric Immobilization of Liposomes in Chromatographic Gel Beads and Incorporation of Integral Membrane Proteins into Their Lipid Bilayers

Interactions between water-soluble substances and immobilized liposomes or proteoliposomes can be analyzed by chromatographic procedures. Methods were therefore developed to sterically immobilize large amounts of liposomes in gel beads. First, freeze-thawing of moist Sephacryl S-1000 beads mixed with egg yolk phospholipid liposomes of 200-300 mM lipid concentration immobilized liposomes representing 60-90 μmol lipid with an internal volume of 60-150 μl per milliliter gel bed. The same procedure with freeze-dried beads immobilized liposomes representing more lipid but with lower internal volumes. Other lipid mixtures could also be used. Second, mixtures of Sephacryl S-1000 beads and liposomes were freeze-dried and rehydrated. Large amounts of liposomes became immobilized even with dilute liposome suspensions. Third, a mixture of diethylether-dried Sephacryl S-1000 beads and an emulsion of an aqueous solution in diethylether with lipids was subjected to reverse-phase evaporation. Liposomes of high specific internal volume became immobilized. Integral human red cell membrane proteins could be incorporated into the liposomal lipid bilayers. Upon mixing octyl glucoside-solubilized proteins with moist beads containing immobilized liposomes, up to 0.4 mg protein became incorporated per milliliter gel bed (yield 34%). Proteins could also be incorporated during immobilization by reverse-phase evaporation. Detergents could be removed efficiently from immobilized (proteo)liposomes in a gel bed.

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid.

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.

Reconstitution of membrane proteins: sequential incorporation of integral membrane proteins into preformed lipid bilayers.

Several integral membrane proteins can be inserted sequentially into preformed unilamellar vesicles (ULV's) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol in a gel phase. Thus, proteoliposomes of DMPC, cholesterol, and bacteriorhodopsin from Halobacterium halobium rapidly incorporate UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase from beef heart mitochondria, and additional bacteriorhodopsin, added sequentially. This process of spontaneous incorporation can be regulated to produce complex artificial membranes that contain phospholipids and proteins at ratios (mol/mol) equivalent to what is found in biological membranes. The ability of the lipid-protein bilayers to incorporate additional integral membrane proteins is not affected by annealing of the proteoliposomes at 37 degrees C nor by the order of addition of the proteins. Bacteriorhodopsin-containing vesicles formed by the sequential addition of integral membrane proteins demonstrate light-driven proton pumping. Therefore, they have retained a vesicular structure. Vesicles containing one or two different proteins will fuse with each other at 21 degrees C or with ULV's devoid of proteins. Incorporation of bacteriorhodopsin or UDPglucuronosyltransferase into proteoliposomes containing DMPC, with or without cholesterol as impurity, also occurs above the phase transition for DMPC. The presence of a protein in a liquid-crystalline bilayer provides the necessary condition for promoting the spontaneous incorporation of other membrane proteins into preformed bilayers.