Lipid Bilayer Arrays Research Articles

Structural Characterization of Nanoparticle-Supported Lipid Bilayer Arrays by Grazing Incidence X-ray and Neutron Scattering

Arrays of nanoparticle-supported lipid bilayers (nanoSLB) are lipid-coated nanopatterned interfaces that provide a platform to study curved model biological membranes using surface-sensitive techniques. We combined scattering techniques with direct imaging, to gain access to sub-nanometer scale structural information on stable nanoparticle monolayers assembled on silicon crystals in a noncovalent manner using a Langmuir-Schaefer deposition. The structure of supported lipid bilayers formed on the nanoparticle arrays via vesicle fusion was investigated using a combination of grazing incidence X-ray and neutron scattering techniques complemented by fluorescence microscopy imaging. Ordered nanoparticle assemblies were shown to be suitable and stable substrates for the formation of curved and fluid lipid bilayers that retained lateral mobility, as shown by fluorescence recovery after photobleaching and quartz crystal microbalance measurements. Neutron reflectometry revealed the formation of high-coverage lipid bilayers around the spherical particles together with a flat lipid bilayer on the substrate below the nanoparticles. The presence of coexisting flat and curved supported lipid bilayers on the same substrate, combined with the sub-nanometer accuracy and isotopic sensitivity of grazing incidence neutron scattering, provides a promising novel approach to investigate curvature-dependent membrane phenomena on supported lipid bilayers.

Encapsulated droplet interface bilayers as a platform for high-throughput membrane studies.

Whilst it is highly desirable to produce artificial lipid bilayer arrays allowing for systematic high-content screening of membrane conditions, it remains a challenge due to the combined requirements of scaled membrane production, simple measurement access, and independent control over individual bilayer experimental conditions. Here, droplet bilayers encapsulated within a hydrogel shell are output individually into multi-well plates for simple, arrayed quantitative measurements. The afforded experimental throughput is used to conduct a 2D concentration screen characterising the synergistic pore-forming peptides Magainin2 and PGLa. Maximal enhanced activity is revealed at equimolar peptide concentrations via a membrane dye leakage assay, a finding consistent with models proposed from NMR data. The versatility of the platform is demonstrated by performing in situ electrophysiology, revealing low conductance pore activity (∼15 to 20 pA with 4.5 pA sub-states). In conclusion, this array platform addresses the aforementioned challenges and provides new and flexible opportunities for high-throughput membrane studies. Furthermore, the ability to engineer droplet networks within each construct paves the way for “lab-in-a-capsule” approaches accommodating multiple assays per construct and allowing for communicative reaction pathways.

Rapid and Resilient Detection of Toxin Pore Formation Using a Lipid Bilayer Array.

An artificial cell membrane is applied to study the pore formation mechanisms of bacterial pore-forming toxins for therapeutic applications. Electrical monitoring of ionic current across the membrane provides information on the pore formation process of toxins at the single pore level, as well as the pore characteristics such as dimensions and ionic selectivity. However, the efficiency of pore formation detection largely depends on the encounter probability of toxin to the membrane and the fragility of the membrane. This study presents a bilayer lipid membrane array that parallelizes 4 or 16 sets of sensing elements composed of pairs of a membrane and a series electrical resistor. The series resistor prevents current overflow attributed to membrane rupture, and enables current monitoring of the parallelized membranes with a single detector. The array system shortens detection time of a pore-forming protein and improves temporal stability. The current signature represents the states of pore formation and rupture at respective membranes. The developed system will help in understanding the toxic activity of pore-forming toxins.

Functional Characterization of Ion Channels Expressed in Eukaryotic Cell-Free Systems using Lipid Bilayer Arrays

Ion channels are known to be promising drug targets, therefore sensitive and reliable screening assays for ion channel activity are needed for development of new drugs. In this work two different ion channels, a cystic fibrosis transmembrane conductance regulator (CFTR) and a transient receptor potential cation channel, subfamily V (TRPV1)were shown to be expressed in vitro with sufficient yield to record their functional activity after reconstitution into planar bilayer arrays on the Micro Electrode Cavity Array (MECA) chip. Eukaryotic cell free expression system enabled correct folding and co-translational integration of synthesized membrane proteins into endogenous endoplasmic reticulum derived vesicles named microsomes as well as a posttranslational phosphorylation of CFTR. For the functional characterization of the in vitro expressed ion channels the collected microsomal membranes were either directly fused to planar lipid bilayers or reconstituted after the solubilization in detergent and subsequent relipidation in proteoliposomes of defined composition. The activity from the single ion channels inserted in different bilayers on the MECA chip was recorded in parallel. The in vitro expressed TRPV1 was shown to display typical single-channel kinetic behavior with steep channel activation above 40 °C as described for the protein isolated from cellular membrane preparations. In vitro synthesized CFTR showed typical conductance values, as well as ATP-dependent activation pattern previously described by the patch-clamp technique. Production of functional mammalian ion channels in eukaryotic cell-free systems together with the possibility of reliable functional activity screening using artificial membrane arrays have a potential to improve drug development pipelines for the search of new inhibitors of ion channel activity.

Ruggedized Chip-Based Lipid Bilayer Arrays with Permeable multi-MAC Support

Planar lipid bilayers play a central role in nanopore sensing and ion-channel electrophysiology. However, bilayer fragility often limits their application. Previously, we enhanced the mechanical properties of planar lipid bilayers with a single-layered, molecularly thin, minimal actin cortex (MAC). To further increase bilayer robustness, we are developing multiple layered MAC structures (multi-MACs) that can be chemically cross-linked to arbitrary thicknesses, without blocking diffusive access to the outer leaflet of the bilayer. In this work, we describe the multi-MAC formation process in the context of a bilayer array on a fluidic chip, as well as on glass supported bilayers. We report on permeability tests at both the single-molecule and macroscopic levels that show multi-MACs do not significantly block access to the bilayer. We discuss how permeability is achieved by controlling the openness of the F-actin network, which can be tailored using the density of linkage sites, the electrostatic interactions between linkers and filaments, and specific F-actin growth and deposition techniques. We also present results from single-molecule electrical and optical measurements that characterize the multi-MAC structure, permeability, and the approximate pore size of the multi-layered network.

Enhancement of membrane protein reconstitution on 3D free-standing lipid bilayer array in a microfluidic channel

The bio-sensory organs of living creatures have evolved to have the best sensing performance. They have 3-dimensional protrusions that have large surface areas to accommodate a large number of membrane proteins such as ion channels and G-protein coupled receptors, resulting in high sensitivity and specificity to target molecules. From the perspective of mimicking this system, BLM, which has been used extensively as a platform for a single nanopore-based sensing systems, has some limitations, i.e., some residual solvent, low mechanical stability, small surface area for appropriate stability, and difficulty in high-throughput fabrication. Herein, to eliminate these limitations, a solvent-free, size-controllable, 3-dimensional free-standing lipid bilayer (3DFLB) structure array with high stability (∼130 h) and high density (∼300,000 cm−2) is proposed, and its structural advantages for efficient and rapid protein reconstitution, compared to BLM, is demonstrated by human 5-HT3A receptor assay as well as α-hemolysin assay. A continuous process of 3DFLB array fabrication, 5-HT3A reconstitution, and 5-HT detections in a microfluidic channel proves the applicability of the proposed structures as a highly-sensitive sensing platform mimicking bio-sensory organs.

Array of Freestanding Planar Lipid Bilayers for Parallel Optical and Electrical Recordings

Here we report on the design and validation of an array platform for parallel high-resolution electrophysiological and optical recordings. The recording chip is based on a SU-8 coated glass slide (ca. 200 μm thickness) containing 4 cavities with individual ring-shaped Ag/AgCl-microelectrodes as well as a common ground electrode. Four suspended lipid bilayers can be self-assembled on the chip surface over the cavities. The electrodes are connected to a multichannel amplifier capable of simultaneous electrical recording with high-bandwidth and low noise (<1pA [email protected]). The ring shape of the electrodes creates an optical window allowing access with a high-NA oil or water-immersion objective on an inverted microscope. This allows for e.g. single-molecule fluorescence measurements from one of the four bilayers using time-correlated-single-photon-counting (TCSPC). Fluorescently labeled alpha-hemolysin for optical and electrical measurements was expressed in-vitro and purified. BODIPY at position 131 was incorporated during the translation in a site-directed manner via stop or four-base codon suppression using pre-charged tRNAs. A series of characterization and validation experiments performed with the system include: (1)Studies of diffusion rates of fluorescently labelled Hemolysin and lipid molecules in the bilayer using fluorescence correlation spectroscopy (FCS); (2)Monitoring of alpha-hemolysin membrane insertion using fluorescence lifetime imaging (FLIM) and (3)Visualization of fusion of labeled proteoliposomes resulting in membrane channel insertion. In the two latter cases, changes in fluorescence intensity were monitored simultaneously with alterations in membrane conductance. This system allows for rapid membrane array formation and by providing ready combination of fluorescence microscopy with high-resolution electrophysiology, enables correlated optical and electrical characterization and monitoring of the interactions of channel forming proteins in artificial lipid bilayers on a single-molecule level. The technology can be further implemented for studies of membrane protein folding and mobility as well as of membrane transport.

Tightly Sealed 3D Lipid Structure Monolithically Generated on Transparent SU-8 Microwell Arrays for Biosensor Applications

Artificial lipid membranes are excellent candidates for new biosensing platforms because their structures are similar to cell membranes and it is relatively easy to modify the composition of the membrane. The freestanding structure is preferable for this purpose because of the more manageable reconstitution of the membrane protein. Therefore, most of the lipid membranes for biosensing are based on two-dimensional structures that are fixed on a solid substrate (unlike floating liposomes) even though they have some disadvantages, such as low stability, small surface area, and potential retention of solvent in the membrane. In this paper, three-dimensional freestanding lipid bilayer (3D FLB) arrays were fabricated uniformly on SU-8 microwells without any toxic solvent. The 3D FLBs have better stability and larger surface area due to their cell-like structure. In order to improve the sealing characteristics of the 3D FLBs, the applied frequency of the ac field was controlled during the electroformation. The 3D FLBs were observed through transparent SU-8 microwell arrays using confocal microscopy and demonstrated perfect sealing until 5.5 days after the electroformation at more than 1 kHz. Also, the details of the sealing of a fixed 3D freestanding lipid structure were discussed for the first time. The unilamellarity and biofunctionality of the 3D FLBs were verified by a transport protein (α-hemolysin) assay.

Development of Lipid Bilayer Array Device for Biological Nanopore Recordings

Development of Lipid Bilayer Array Device for Biological Nanopore Recordings

Selective protein recognition in supported lipid bilayer arrays by tailored, dual-mode deep cavitand hosts.

Self-folding deep cavitands with variably functionalized upper rims are able to selectively immobilize proteins at a biomimetic supported lipid bilayer surface. The immobilization process takes advantage of the dual-mode binding capabilities of the hosts, combining a defined binding pocket with upper rim charged/H-bonding groups. A variety of proteins can be selectively immobilized at the bilayer interface, either via complementary charge/H-bonding interactions, cavity-based molecular recognition, or a combination of both. The immobilization process can be used to bind unmodified native proteins, epitopes for bioadhesion, or proteins covalently modified with suitable RNMe3+ binding "handles" and charged groups that can either match or mismatch with the cavitand rim. The immobilization process can be monitored in real time using surface plasmon resonance (SPR) spectroscopy, and applied to the construction of cavitand:lipid arrays using the hosts and trehalose vitrified phospholipid vesicles. The selective, dual-mode protein recognition is maintained in the arrays, and can be visualized using SPR imaging.

Micropatterning of Phase-Segregated Supported Lipid Bilayers and Binary Lipid Phases through Polymer Stencil Lift-Off.

Supported lipid bilayers (SLBs) provide an excellent model system for studying structural and functional characteristics of biomembranes. Patterning model membranes on solid supports has elicited much interest because lipid bilayer arrays at cellular or subcellular scales provide attractive platforms for reconstituting tissue-like conditions for cell culture, and for creating simplified physiological environments to study biological processes. Phase-segregated SLB patterns can be especially useful for such studies, as the selective functionalization of the lipid phases with different lipids, receptors, or proteins can be achieved to mimic the key features of plasma membrane. However, it remains challenging to pattern phase-segregated lipid bilayers and to spatially control the lipid phases at the micron scale. Current methods to achieve this involve multiple surface modification and patterning steps, elaborate techniques such as microfluidic, microcontact printing, or electrochemical control, among others. To overcome the complexity in producing phase-segregated patterns, we have developed simple and rapid strategies to pattern SLBs with phase separation utilizing the polymer stencil lift-off (PSLO) technique. PSLO is a powerful technique for SLB patterning, since it allows the faithful pattern transfer of micron-sized lipid domains onto solid surfaces under aqueous conditions, which eliminates the need for controlled humidity and reduces the risk of bilayer disruption through drying. By integrating postetching substrate cleaning and a blocking treatment, well-defined homogeneous and phase-segregated SLB patterns were achieved with lipid mobility that matches that of SLBs formed on clean SiO2 wafer substrates. A two-step incubation method was also developed for patterning binary lipid phases, which allowed precise control of their position and geometries. The created phase-segregated SLB patterns were used to study lipid phase behavior within confined areas, and quantitative analysis showed that smaller pattern sizes resulted in smaller gel phase domains, which also covered a smaller fraction of the total patterned SLB area. This was attributed to the decreased mobility of the bottom leaflet of the SLB, which lies in close proximity to the substrate, and the resulting hindered exchange of lipid molecules between the bottom and upper leaflets through the SLB boundary. By further integration with functional groups, the phase-segregated lipid bilayer patterns might find relevant application in tissue engineering, biophysical studies of biomolecular and cellular interactions, and biosensing platforms.

Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array.

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.

HERG Ion Channel Activity Measured in Planar Lipid Bilayer Arrays: Reconstitution from Eukaryotic Cell-Free Expression System and Cellular Membrane Preparations

Ion channels are important pharmacological targets. Therefore there is an urgent need for new technologies for high throughput ion channel activity monitoring and pharmacology screening capable to substitute currently used labor-intensive and expensive cell-based electrophysiological assays. Ion channels reconstituted into artificial membrane arrays may serve as such test system offering a substantial simplification as well as great flexibility in controlling the ion channel environment and experimental conditions.Human ether-a-go-go-related gene (hERG), a voltage dependent potassium channel is a major target of pharmacology safety screening due to its critical importance in heart function. Here we report on parallel high-resolution electrophysiological recordings from different hERG-containing preparations reconstituted into bilayer lipid membranes on the micro electrode cavity array (MECA) chip.Two different sources of hERG channels have been investigated: (1) Membrane preparations from a hERG-expressing HEK293 cell line, and (2) Vesicles collected after cell-free expression of the hERG channels with their co-translational integration into microsomes or liposomes. The hERG-containing vesicles were fused with lipid bilayers preformed on the MECA chip. For both preparations fluctuating currents of different amplitudes have been recorded following the fusion. Single channel and channel ensemble currents depending on the vesicle preparation have been characterized. The recorded currents were shown to be partially inhibited by the hERG specific blockers E4031 and astemizole.High channel expression level as well as optimized vesicles preparation and fusion conditions were shown to be crucial for the reproducible channel recordings in the artificial membrane system. Addition of charged lipids (POPG/DOTAP) to the liposomes and acceptor membranes was found to facilitate vesicle fusion and improve the measurements success rate. The presented technology has a potential for a high throughput ion channels pharmacology screening in general.

Supported lipid bilayer membrane arrays on micro-patterned ITO electrodes

Lipid bilayer arrays were formed on micropatterned ITO electrodes. With this bilayer array platform both the fluorescence microscopy and electrochemical detection can be realized to explore the biophysical properties of cell membrane.

Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles

Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.

On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging.

The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents.

Nanoparticle-Induced Membrane Pore Formation Studied with Lipid Bilayer Arrays

Nanoparticles are present environmentally as byproducts of industrial processes and in a wide range of consumer goods. Although toxicity studies of nanoparticles and other nanomaterials have only begun relatively recently, a diverse range of nanoparticles has already been shown to be toxic. Nanoparticle cytotoxicity assays provide little information about the mechanisms of nanoparticle toxicity, which are potentially complex and are not likely the same for diverse nanoparticle species. For this reason, artificial lipid bilayer platforms have begun to be used as model systems for controlled studies allowing variation of experimental parameters not possible with cellular studies, such as membrane and solution composition. Unfortunately, in some cases the low throughput characteristic of lipid bilayer experimentation can limit the experimental scope. We have recently described a lipid bilayer array platform with simultaneous bilayer formation and measurement over a 32-element array with ∼80% yield and no operator input following fluid addition.[1] The platform is modular and allows rapid cycling of the apparatus for repeated measurement. We have used this platform to measure the interactions of aminated and carboxylated polystyrene nanoparticles and a range metal oxide nanoparticle species with lipid bilayers in a wide variety of experimental conditions, including nanoparticle concentration, bilayer composition, bilayer charge, presence of serum protein, solution ionic strength, and pH. The array format permitted several thousand bilayers to be measured in total with sufficient redundancy to give statistical significance to measured results. Detailed analysis of the electrical measurements shows pore formation that is dependent on electric field, ionic strength, and nanoparticle species.1. “Lipid bilayer arrays cyclically formed and measured,” Bin Lu, Gayane Kocharyan and Jacob J. Schmidt, Biotechnology Journal 9, 446-451 (2014)

Nanoparticle–lipid bilayer interactions studied with lipid bilayer arrays

The widespread environmental presence and commercial use of nanoparticles have raised significant health concerns as a result of many in vitro and in vivo assays indicating toxicity of a wide range of nanoparticle species. Many of these assays have identified the ability of nanoparticles to damage cell membranes. These interactions can be studied in detail using artificial lipid bilayers, which can provide insight into the nature of the particle-membrane interaction through variation of membrane and solution properties not possible with cell-based assays. However, the scope of these studies can be limited because of the low throughput characteristic of lipid bilayer platforms. We have recently described an easy to use, parallel lipid bilayer platform which we have used to electrically investigate the activity of 60 nm diameter amine and carboxyl modified polystyrene nanoparticles (NH2-NP and COOH-NP) with over 1000 lipid bilayers while varying lipid composition, bilayer charge, ionic strength, pH, voltage, serum, particle concentration, and particle charge. Our results confirm recent studies finding activity of NH2-NP but not COOH-NP. Detailed analysis shows that NH2-NP formed pores 0.3-2.3 nm in radius, dependent on bilayer and solution composition. These interactions appear to be electrostatic, as they are regulated by NH2-NP surface charge, solution ionic strength, and bilayer charge. The ability to rapidly measure a large number of nanoparticle and membrane parameters indicates strong potential of this bilayer array platform for additional nanoparticle bilayer studies.

Rapid Activity Screening of Ion Channels Expressed in Cell-Free Systems Using a Lipid Bilayer Array Pcb-Device

Cell-free expression is a rapidly developing tool for the efficient production of membrane proteins with enormous potential. Here we report on the design of a setup for parallel high-resolution electrophysiological recordings allowing for the screening of functional activity of ion channels synthesized in-vitro in various lipid environments.The developed recording chamber is based on a SU-8 coated printed circuit board containing 4 cavities (50µm) with individual microelectrodes (Micro-Electrode-Cavity-Array (MECA) as well as a common ground electrode. 4 suspended lipid bilayers can be self-assembled on the nonpolar chip surface from phospholipids in organic solvent. The MECA-PCB is connected to a multichannel amplifier capable of simultaneous recording of electrical activity from the ion channels functionally reconstituted into the bilayers with high-bandwidth and low noise (<1 pA rms ≅ 10 kHz).To test the capability of the MECA-PCB-chip, the tetrameric potassium channel KcsA was expressed in-vitro with co-translational insertion into supplied liposomes containing di-myristoyl-glycerophosphocholine or asolectin. The collected proteoliposomes were extruded through 100 nm polycarbonate filter and fused with preformed bilayers. Activity from the single channels inserted in different bilayers was recorded in parallel under identical conditions. We show thatKcsA expressed in-vitro displays the typical single-channel kinetic behavior and sensitivity to blockers (Ba2+) described for the protein isolated from cellular membrane preparations. Channel open times and open probability are shown to be dependent on lipid environment with negatively-charged phospholipids stabilizing the open channel conformation.The combination of the cell-free protein expression system and MECA-PCB-chip electrophysiology allows for functional characterization of the synthesized ion channel within several hours starting from the DNA template. The current PCB-chip design could be easily adapted for higher throughput and automated bilayer formation as recently reported1.1Del Rio Martinez et.al. Small 2014.

Spontaneous transmembrane helix insertion thermodynamically mimics translocon-guided insertion.

The favorable transfer free energy for a transmembrane (TM) α-helix between the aqueous phase and lipid bilayer underlies the stability of membrane proteins. However, the connection between the energetics and process of membrane protein assembly by the Sec61/SecY translocon complex in vivo is not clear. Here, we directly determine the partitioning free energies of a family of designed peptides using three independent approaches: an experimental microsomal Sec61 translocon assay, a biophysical (spectroscopic) characterization of peptide insertion into hydrated planar lipid bilayer arrays, and an unbiased atomic-detail equilibrium folding-partitioning molecular dynamics simulation. Remarkably, the measured free energies of insertion are quantitatively similar for all three approaches. The molecular dynamics simulations show that TM helix insertion involves equilibrium with the membrane interface, suggesting that the interface may play a role in translocon-guided insertion.