Oligomerization and aggregation of NAP-22 with several metal ions.

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Controlling Physical and Biochemical Parameters of Actin Nucleation Using a Patterned Model Lipid Membrane.

Self-assembly of nanoscale actin cytoskeletal proteins into filamentous networks requires organizing actin nucleation areas on the plasma membrane through recruiting actin nucleators and nucleation-promoting factors (NPFs) to the areas. To investigate impacts of the nucleation geometry on actin network assembly, we localized NPF or nucleator on defined micropatterns of laterally mobile lipid bilayers confined in a framework of a polymerized lipid bilayer. We demonstrated that actin network assembly in purified protein mixtures was confined on NPF- or nucleator-localized fluid bilayers. By controlling the shape and size of nucleation areas as well as the density and types of localized NPFs and nucleators, we showed that these parameters regulate actin network architectures. Actin network assembly in Xenopus egg extracts was also spatially controlled by patterning bilayers containing phosphatidylinositol 4,5-bisphoshate (PI(4,5)P2), an essential lipid signaling mediator. Therefore, the system provides a promising platform to investigate the physical and biochemical principles for actin network assembly.

Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo "self-quenching" of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 µm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Retarded Diffusion and Confinement of Membrane-Bound Molecules in a Patterned Hybrid Membrane of Phospholipid Bilayers and Monolayers.

The biological membrane is a complex two-dimensional fluid, in which various molecular interactions regulate the lateral diffusion of membrane-associated molecules. Pinning of membrane proteins or lipids by extra-membrane proteins impedes the diffusion. In addition, coupling between two monolayer leaflets within a phospholipid bilayer via interdigitation plays important roles, though this effect remains elusive. Here, we fabricate a substrate-supported model membrane with patterned bilayer/monolayer regions to explore the influences of interleaflet coupling. A patterned monolayer of polymerized diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), was lithographically generated and used to form patterned lipid bilayers and monolayers. A phospholipid monolayer was formed on top of the polymerized monolayer. The bilayer/monolayer hybrid membrane was continuous and fluid, but lateral diffusion in the monolayer region was significantly retarded, suggesting the influences of interleaflet coupling. We reconstituted photoreceptor rhodopsin (Rh) and G-protein transducin (Gt) as model transmembrane and peripheral proteins. Rh diffused laterally only in the bilayer region, whereas Gt diffused in both bilayer and monolayer regions. The patterned hybrid bilayer/monolayer membrane reproduces the retarded diffusion and confinement of membrane-bound molecules in a controlled manner and provides insight into the physicochemical and functional roles of semipermeable corrals in the cell membrane.

Enhancing the spectral range of plant and bacterial light-harvesting pigment-protein complexes with various synthetic chromophores incorporated into lipid vesicles.

The Light-Harvesting (LH) pigment-protein complexes found in photosynthetic organisms have the role of absorbing solar energy with high efficiency and transferring it to reaction centre complexes. LH complexes contain a suite of pigments that each absorb light at specific wavelengths, however, the natural combinations of pigments within any one protein complex do not cover the full range of solar radiation. Here, we provide an in-depth comparison of the relative effectiveness of five different organic "dye" molecules (Texas Red, ATTO, Cy7, DiI, DiR) for enhancing the absorption range of two different LH membrane protein complexes (the major LHCII from plants and LH2 from purple phototrophic bacteria). Proteoliposomes were self-assembled from defined mixtures of lipids, proteins and dye molecules and their optical properties were quantified by absorption and fluorescence spectroscopy. Both lipid-linked dyes and alternative lipophilic dyes were found to be effective excitation energy donors to LH protein complexes, without the need for direct chemical or generic modification of the proteins. The Förster theory parameters (e.g., spectral overlap) were compared between each donor-acceptor combination and found to be good predictors of an effective dye-protein combination. At the highest dye-to-protein ratios tested (over 20:1), the effective absorption strength integrated over the full spectral range was increased to ∼180% of its natural level for both LH complexes. Lipophilic dyes could be inserted into pre-formed membranes although their effectiveness was found to depend upon favourable physicochemical interactions. Finally, we demonstrated that these dyes can also be effective at increasing the spectral range of surface-supported models of photosynthetic membranes, using fluorescence microscopy. The results of this work provide insight into the utility of self-assembled lipid membranes and the great flexibility of LH complexes for interacting with different dyes.

Nanofluidic Model Membrane for the Single-Molecule Observation of Membrane Proteins.

Membrane proteins play essential roles in the cell, and they constitute one of the most important targets of drugs. Studying membrane proteins in a controlled model membrane environment can provide unambiguous, quantitative information on their molecular properties and functions. However, reconstituting membrane proteins in a model system poses formidable technological challenges. Here, we developed a novel model membrane platform for highly sensitive observation of membrane proteins by combining a micropatterned lipid membrane and a nanofluidic channel. A micropatterned model membrane was generated by lithographically integrating a polymerized lipid bilayer and a natural (fluid) lipid bilayer. A nanofluidic channel having a defined thickness was formed between the fluid bilayer and a polydimethylsiloxane (PDMS) slab by attaching the polymeric bilayer and PDMS slab using an adhesion layer composed of silica nanoparticles that are coated with a biocompatible polymer brush. As we reconstituted rhodopsin (Rh), a G-protein-coupled receptor (GPCR), from a detergent-solubilized state into the fluid bilayer, only successfully reconstituted Rh molecules diffused laterally in the lipid bilayer and migrated into the nanogap junction, where they could be observed with a vastly improved signal-to-background ratio. The nanogap junction effectively separates the sites of reconstitution and observation and provides a novel platform for studying the molecular properties and functions of membrane proteins at the single-molecular level.

Functional Reconstitution of Dopamine D2 Receptor into a Supported Model Membrane in a Nanometric Confinement.

Dopamine D2 receptor (D2R), a G-protein-coupled receptor (GPCR), plays critical roles in neural functions and represents the target for a wide variety of drugs used to treat neurological diseases. However, its fundamental physicochemical properties, such as dimerization and affinity to different lipid environments, remain unknown. Here, reconstitution and characterization of D2R in a supported model membrane in nanometric confinement are reported. D2R is expressed in Chinese hamster ovary (CHO) cells and transferred into the supported model membrane as cell membrane blebs. D2R molecules are reconstituted with an elevated density in the cleft between the substrate and poly(dimethylsiloxane) (PDMS) elastomer. Reconstituted D2R retains the physiological functions, as evaluated from its binding to an antagonist and dimerization lifetime. The transient dimer formation of D2R, similar to the live cell, suggests that it is an innate property that does not depend on the cellular structures such as actin filaments. Although the mechanism of this unique reconstitution process is currently not fully understood, the finding points to a new possibility of using a nanometric space (<100 nm thick) as a platform for reconstituting and studying membrane proteins under the quasi-physiological conditions, which are difficult to be created by other methods.

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light-Harvesting Proteins.

Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.

Substrate-supported Model Membrane as a Versatile Analytical/Biosensing Platform.

Biological membranes composed of a lipid bilayer and associated proteins work as a platform for highly selective and sensitive detection in nature. Substrate-supported lipid bilayers (SLBs) are a model system of the biological membrane that are mechanically stable, accessible to highly sensitive analytical techniques, and amenable to micro-fabrication, such as patterning. The surface of SLBs can effectively suppress the non-specific binding of proteins, and enhance selective detection by specific interactions. These features render SLBs highly attractive for the development of devices that utilize artificially mimicked cellular functions. Furthermore, SLBs can be combined with nanoscopic spaces, such as nano-channels and nano-pores, that can reduce the detection volume and suppress the non-specific background noise, enhancing the signal-to-background noise (S/B) ratio. SLBs therefore provide promising platforms for a wide range of biomedical and environmental analyses.

The effects of phospholipids and fatty acids on the oligomer formation of NAP-22.

Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM fraction of the neuronal cell membrane. Previous studies showed tight binding activity of NAP-22 to acidic membrane lipids and the self-interaction of NAP-22, i.e., oligomerization. In this study, the effect of various phospholipids, lysophospholipids and fatty acids on the oligomerization of NAP-22 was studied through SDS-PAGE after chemical cross-linking and electron microscopic observation. High molecular mass oligomers were detected by SDS-PAGE after incubation in solutions containing over 20 mM NaCl at pH 6.5-8.5, even in the absence of lipid addition, and the addition of Ca2+/calmodulin abolished oligomerization. Higher molecular mass oligomer formation after incubation with acidic phospholipids was detected with gradient SDS-PAGE. Much higher mass oligomers were detected in the presence of polyunsaturated fatty acids. Electron microscopic analysis of the samples after SDS treatment showed tangled rope-like structures. Liposome-bound NAP-22 showed small oval or annular structures after cross-linking and SDS treatment. These oligomers were suggested to make the tangled rope-like structures, for annular structures of the same size were observed in the structure. These results suggest the participation of NAP-22 to liquid-liquid phase separation through oligomerization.

Photosynthetic Model Membranes of Natural Plant Thylakoid Embedded in a Patterned Polymeric Lipid Bilayer.

Thylakoid membranes in the chloroplast of plants, algae, and cyanobacteria are the powerhouse of photosynthesis, capturing solar energy and converting it into chemical energy. Although their structures and functions have been extensively studied, the intrinsically heterogeneous and dynamic nature of the membrane structures is still not fully understood. Investigating native thylakoid membranes in vivo is difficult due to their small size and limited external access to the chloroplast interior, while the bottom-up approaches based on model systems have been hampered by the sheer complexity of the native membrane. Here, we try to fill the gap by reconstituting the whole thylakoid membrane into a patterned substrate-supported planer bilayer. A mixture of thylakoid membrane purified from spinach leaves and synthetic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles spontaneously formed a laterally continuous and fluid two-dimensional (2D) membrane in the scaffold of the patterned polymeric bilayer. Chlorophyll fluorescence arising from photosystem II (PSII) recovered after photobleaching, suggesting that the membrane components are laterally mobile. The reversible changes of chlorophyll fluorescence in the presence of the electron acceptors and/or inhibitors indicated that the electron transfer activity of PSII was retained. Furthermore, we confirmed the electron transfer activity of photosystem I (PSI) by observing the generation of nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of water-soluble ferredoxin and ferredoxin-NADP+ reductase. The lateral mobility of membrane-bound molecules and the functional reconstitution of major photosystems provide evidence that our hybrid thylakoid membranes could be an excellent experimental platform to study the 2D molecular organization and machinery of photosynthesis.

Affinity of rhodopsin to raft enables the aligned oligomer formation from dimers: Coarse-grained molecular dynamics simulation of disk membranes.

The visual photopigment protein rhodopsin (Rh) is a typical G protein-coupled receptor (GPCR) that initiates the phototransduction cascade in retinal disk membrane of rod-photoreceptor cells. Rh molecule has a tendency to form dimer, and the dimer tends to form rows, which is suggested to heighten phototransduction efficiency in single-photon regime. In addition, the dimerization confers Rh an affinity for lipid raft, i.e. raftophilicity. However, the mechanism by which Rh-dimer raftophilicity contributes to the organization of the higher order structure remains unknown. In this study, we performed coarse-grained molecular dynamics simulations of a disk membrane model containing unsaturated lipids, saturated lipids with cholesterol, and Rh-dimers. We described the Rh-dimers by two-dimensional particle populations where the palmitoyl moieties of each Rh exhibits raftophilicity. We simulated the structuring of Rh in a disk for two types of Rh-dimer, i.e., the most and second most stable Rh dimers, which exposes the raftophilic regions at the dimerization-interface (H1/H8 dimer) and two edges away from the interface (H4/H5 dimer), respectively. Our simulations revealed that only the H1/H8 dimer could form a row structure. A small number of raftophilic lipids recruited to and intercalated in a narrow space between H1/H8 dimers stabilize the side-by-side interaction between dimers in a row. Our results implicate that the nano-sized lipid raft domains act as a "glue" to organize the long row structures of Rh-dimers.

Self-Spreading of Phospholipid Bilayer in a Patterned Framework of Polymeric Bilayer.

Phospholipid bilayers spontaneously spread on a hydrophilic substrate such as glass in aqueous solution due to the energetic gain of surface wetting. This process (self-spreading) was utilized to form a patterned model biological membrane containing reconstituted membrane proteins. A mechanically stable framework of a polymerized lipid bilayer was first generated by the lithographic polymerization of a diacetylene phospholipid. Then, natural lipid membranes (fluid bilayers) were introduced into the channels between polymeric bilayers by the self-spreading from a phospholipid reservoir. The spreading velocity could be fitted into a slope of -0.5 in a double logarithmic plot versus time due to the balance between the spreading force and resistive drag. The preformed polymeric bilayer accelerated the spreading by the energetic gain of covering hydrophobic edges with a lipid bilayer. At the same time, the domains of the polymeric bilayer obstructed spreading, and the spreading velocity linearly decreased with their fractional coverage. Above the critical coverage of ca. 50%, self-spreading was completely blocked (percolation threshold) and the fluid bilayer was confined in the polymer-free regions. Nonspecific adsorption of lipids onto the surface of polymeric bilayers was negligible, which enabled a heightened signal-to-background ratio in the reconstitution and observation of membrane proteins. Self-spread bilayers had a higher density of lipids than those formed by the spontaneous rupture of vesicles (vesicle fusion), presumably due to the continual supply of lipid molecules from the reservoir. These features give the self-spreading important advantages for preparing patterned model membranes with reconstituted membrane proteins.

Raftophilic rhodopsin-clusters offer stochastic platforms for G protein signalling in retinal discs.

Rhodopsin is a G protein-coupled receptor (GPCR) that initiates the phototransduction cascade in retinal disc membrane. Recent studies have suggested that rhodopsin forms highly ordered rows of dimers responsible for single-photon detection by rod photoreceptors. Dimerization is also known to confer to rhodopsin a high affinity for ordered lipids (raftophilicity). However, the role of rhodopsin organization and its raftophilicity in phototransduction remains obscure, owing to the lack of direct observation of rhodopsin dynamics and distribution in native discs. Here, we explore the single-molecule and semi-multimolecule behaviour of rhodopsin in native discs. Rhodopsin forms transient meso-scale clusters, even in darkness, which are loosely confined to the disc centre. Cognate G protein transducin co-distributes with rhodopsin, and exhibits lateral translocation to the disc periphery upon activation. We demonstrate that rhodopsin offers inherently distributed and stochastic platforms for G protein signalling by self-organizing raftophilic clusters, which continually repeat generation/extinction in the disc membrane.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

Nanofluidic Biosensor Created by Bonding Patterned Model Cell Membrane and Silicone Elastomer with Silica Nanoparticles.

Selective and sensitive detection of specific molecules in a solution containing diverse coexisting molecules is important in many biomedical and environmental applications, including diagnostics and pollutant detection. Here, a nanofluidic biosensor is developed to detect specific target molecules (e.g., toxin proteins) in the presence of nontarget molecules by bonding a patterned model cell membrane and a silicone elastomer (polydimethylsiloxane: PDMS) sheet using surface-modified silica nanoparticles as the adhesive layer. Owing to the uniform size of nanoparticles, a nanometric gap junction is formed between the fluid bilayer and PDMS (nanogap-junction). The thickness of the nanogap-junction is controlled by the size of the silica nanoparticles. Target molecules that specifically bind to the receptor molecules in the fluid bilayer are selectively transported into the nanogap-junction via lateral diffusion through the lipid membrane. A thinner gap formed with smaller nanoparticles can enhance the sensitivity (signal-to-background ratio) more effectively, owing to the suppression of nonspecific penetration of coexisting molecules. Silica nanoparticles also provide excellent mechanical robustness, realizing long-term stability of the gap structure. Nanogap-junction using silica nanoparticles provides a versatile platform for highly selective and sensitive sensing by realizing detection of specific target molecules in a solution containing more concentrated nontarget molecules.

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates.

Hybrid Model Membrane Combining Micropatterned Lipid Bilayer and Hydrophilic Polymer Brush.

Substrate-supported planar lipid bilayers (SPBs) are being utilized as a versatile model system of the biological membrane. However, the proximity between the solid support and membrane limits utility of SPBs for the functional analyses of membrane proteins. Here, we present a model membrane that can enlarge the distance between the substrate surface and the membrane by combining a stable scaffold of polymerized lipid bilayer with a hydrophilic polymer brush. A micropatterned SPB was generated by the lithographic polymerization of diacetylene lipids and subsequent incorporation of natural (fluid) lipid bilayers. Hydrophilic polymer brush of poly-2-methacryloyloxyethyl phosphorylcholine (poly(MPC)) was formed on the surface of polymeric bilayer by the in situ atom transfer radical polymerization (ATRP) in aqueous solution, in the presence of embedded fluid lipid bilayers. A model membrane protein (Haloquadratum walsbyi bacteriorhodopsin: HwBR) could be reconstituted into the polymer brush-supported bilayers with significantly reduced immobile molecules. Furthermore, the polymer brush terminals could be functionalized by successively polymerizing MPC and 2-aminoethyl methacrylate (AMA). The reactive amine moiety of poly(AMA) enables to conjugate a wide range of biological molecules and surfaces to the membrane. The combination of micropatterned bilayer and polymer brush mimics the two- and three-dimensional structures of the biological membrane, providing a platform to assay membrane proteins in a truly biomimetic environment.