The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.
Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.
The analysis of membrane proteins has become of increasing interest for basic and pharmaceutical research in the past 20 years. The development of novel drugs depends on the identification and detailed characterization of new targets, currently being one of the limiting factors. The fact that about 60% of all drug targets are membrane proteins1, makes the development of techniques to elucidate their function most important.
In the past, techniques for the study of electrogenic channels and transporters have been developed in multitude2–4. Non-electrogenic substrates in contrary present a more challenging task. They are however of special interest as prime drug targets, as they control the flux of solutes and nutrients across the cell membrane and function as key receptors in signaling cascades5.
Considerable effort has been put into the development of techniques to study the function of membrane transport proteins6,7. Systems using solid-supported membranes have emerged as most promising tools in this field8–10, including solid supported lipid bilayers, tethered bilayers11,12, microblack lipid membranes13,14 and native vesicle arrays15,16 to name a few. Some of them are even available as commercial setups17,18. Some examples have been published combining the ability to study single membrane proteins in a highly parallel manner14,19, a prerequisite for screening applications. However, these methods rarely bridge from basic research to the industrial environment. The difficulties often lie in the ability of the system to be automatable, the cost-intensive production and/or laborious preparation. An approach overcoming all the above mentioned obstacles is the final aim.
The technique presented here was developed to study membrane channels and transporters in vitro in a controlled environment on the single protein level20–22. Reconstitution of purified membrane proteins into LUVs is much more established than comparable approaches for GUVs23–26 or black lipid membranes27. They can directly be applied to the chip surface, where bilayer formation is taking place via a self-assembly process. The glass-bottom design of the nanoporous chip (Fig. 1) allows for air microscopy, which permits the straightforward automation of the system. In combination with a motorized stage multiple chips can be measured at the same time, with each field of view containing thousands of sealed cavities for analysis.
Figure 1. Design of multiplexed nanopore biochips. A) A silicon-on-insulator (SOI) wafer is structured by reactive-ion etching. Approximately 1,150 individual chips are fabricated from each wafer with identical properties and quality. B) Each chip comprises 250,000 individual microcavities with nano apertures. Scale bar: 200 µm. C) Each cavity is addressable via multi-spectral fluorescence read-out. An intransparent top layer blocks the fluorescent signals from the buffer reservoir, making the biochip compatible with inverted fluorescence microscopes. D) Atomic force microscopy (AFM) imaging reveals evenly arranged pore openings and surface roughness of the silicon dioxide layer of 3.6 nm (n = 40) optimal for vesicle fusion. Scale bar: 5 µm. E) Scanning electron microscopy (SEM) image shows a cross-section through the nanopore allowing access to the femtoliter cavities inside the silicon chip. This figure was reused with permission from 21. Please click here to view a larger version of this figure.
All data analysis is performed using freeware to guarantee unrestricted access for end users. Time series are analyzed using free image processing software and a custom build curve analysis software enabling batch processing and straightforward correlation of large datasets with multiple fluorescent channels and thousands of curves.
The model protein used in this protocol is the mechanosensitive channel of large conductance (MscL) channel protein derived from E. coli. It functions as a valve to release osmotic shock in nature, but was modified in such a way that rationally designed synthetic functionalities can covalently be attached to the channels constriction side. Via charge-repulsion of the covalently bound activator (MTSET) the channel is triggered to open, creating a nano-valve. Small molecules like ions, water, small proteins, but also small fluorophores can permeate through the channel. Here, the protein is used as a model to demonstrate the ability of the system to detect protein-mediated translocation.
1. Preparation of Large Unilamellar Vesicles (LUVs)
2. Protein Reconstitution
3. Activity Assay
4. Size Distribution Measurement of LUVs Using Nano-particle Tracking
5. Chip Holder Preparation
6. Assay Preparation
Note: Nanopore chips glued to a cover glass and separated by an 8-well sticky-slide possess the benefit, that multiple chips can be addressed in a single experimental run, with chips being completely separated from each other.
7. Assay Setup
8. Data Analysis
Pore-spanning membranes can easily be created on the nanostructured chip surface in a self-assembled manner. However the underlying process is still delicate and influenced by many parameters like liposome size, monodispersity of the liposome population, lamellarity, lipid- and salt-concentration and chemical surface properties. Most of these parameters have been carefully characterized and standardized in the above protocol. Other parameters however should be checked during every new preparation, to ensure a successful experiment. These are the liposome size distribution and population dispersity (Fig. 2A). For optimal pore-spanning membrane formation and to avoid liposome intrusion into the cavity space liposome sizes well above the pore diameter are a necessity. Furthermore, monodisperse liposome preparations have been shown to yield best results in membrane spreading and fusion. Another crucial step that has to be checked for every preparation is protein activity after reconstitution. Using a fluorescence-dequenching assay for liposomes reconstituted with MscLG22C, channel opening can be monitored via fluorescence spectroscopy, giving a ratio of liposomes harboring active MscLG22C protein (Fig. 2B).
Once spread to the chip surface, solute translocation of a fluorescent target by MscLG22C upon ligand activation can be followed (Fig. 3B). Engineered MscLG22C channels are closed in their default state, entrapping small fluorescent molecules inside the cavity space. By addition of the chemical modulator MTSET positive charges are introduced to the constriction region of the MscLG22C pore, pushing it open via electrostatic repulsion. The prior entrapped fluorophore evades the cavity space to achieve diffusion equilibrium. The subsequent decrease of fluorescence inside the cavity space can be monitored. This process is highly reproducible, resulting in a homogenous population of efflux events (Fig. 3C). The resulting efflux curves cluster in two distinct populations, demonstrating the ability of the assay to discriminate between single- and multichannel translocation events (Fig. 3D). Furthermore the system is able to follow up to three spectrally well separated dyes in parallel and in real-time, making it possible to record high-content screenings and discriminate precisely and objectively between positive, false positive and negative results. In this study 9.046 sealed cavities were analyzed, of which 8% displayed efflux behavior. Deploying the different fluorescent read-out channels, events can be discriminated into monoexponential efflux events, complex kinetics, lipid intrusions and membrane ruptures (Fig. 3E). Only events that display steady signals for the control solute and the membrane dye are considered for final analysis. Complex kinetics represent efflux events with unsteady control signals and are therefore omitted. Lipid intrusions and membrane ruptures are artifact events intrinsically occurring using pore-spanning lipid bilayer systems. They can be discarded using the control dye signals as mentioned above (Fig. 4).
Figure 2. Size distribution of proteoliposomes and MscL function. A) Vesicles were analyzed by nano-particle tracking analysis. LUV samples were examined before (black trace) and after reconstitution of MscLG22C (red trace). After LUV preparation, a mean particle size of 214 ± 70 nm is achieved. The size is decreased during reconstitution, resulting in proteoliposomes of 158 ± 60 nm in size. B) MscL activity was validated after reconstitution by an efflux assay.28 Self-quenched calcein is released from proteoliposomes through MTSET-mediated opening of the MscL channel, leading to dequenching of the fluorophore (red) and a rapid increase of the fluorescence intensity. Liposomes without protein show no such efflux, even after repeated MTSET addition (black). Subsequent detergent-introduced solubilization (TX-100) of the liposomes releases all previously encapsulated dye. This figure was reused with permission from 21. Please click here to view a larger version of this figure.
Figure 3. Ligand-gated solute translocation by MscL at single-molecule level. A) Engineered MscLG22C channels are closed in their default state. Small solutes such as a fluorophore are unable to pass neither through the channel nor the lipid bilayer. Upon addition of MTSET, an engineered single cysteine of each MscLG22C protomer gets modified, introducing multiple positive charges in the constriction side of the pentameric channel complex. The MscLG22C channel is pushed open by charge repulsion and the entrapped dye can diffuse out of the cavity, leading to a decrease in fluorescence intensity. B) Time traces of MscLG22C opening upon addition of MTSET. Proteoliposomes containing functionally reconstituted MscLG22C were spread on the chip surface. Oy647 (red) was entrapped inside the cavities as translocation substrate. OregonGreen Dextran (70 kDa, green) was added as channel impermeable control substrate to monitor the substrate specificity of the transport event as well as the membrane integrity during the experiment. LUVs were supplemented with DOPEATTO390 (0.1 mol%; purple) to check for lipid contamination inside cavities. Addition of MTSET (3 mM final) at t = 0 sec opens the channel, leading to Oy647 efflux from the cavities, recorded via fluorescence read-out. C) Randomly picked MscLG22C efflux curves demonstrate typical efflux events. D) Rate constants for translocation events (n = 242) cluster into two Gaussian populations, demonstrating that single- and multi-channel transport events can be discriminated. E) High-content screening and statistical analysis of 9,046 sealed chip cavities by triple-color real-time readout. 8% of all analyzed chip cavities show exponential efflux events. Due to spectral decoding (red: translocated solute; green: control solute; violet: lipid), efflux events, complex kinetics, lipid intrusions, and membrane ruptures could be discriminated, thus eliminating false positives. This figure was reused with permission from 21. Please click here to view a larger version of this figure.
Figure 4. Event classes distinguished by three-color detection. Translocated solute (red), control solute (green) and a lipid dye (violet) can be spectrally discriminated allowing for unbiased data selection. A) Sealed cavities harboring no membrane protein show no flux events under the experimental conditions described for the MscL channel recording (see Fig. 3A). The fluorescent readout is stable for all channels. B) Ill-defined suspended lipid bilayers are able to intrude the cavity. C) Membrane rupture results in discontinuous lipid bilayer spanning the nanopore fissures. Thereby, the cavity space is no longer separated from the buffer compartment. Entrapped dyes (red) evade the cavity down the concentration gradient. The control solute (green), previously unable to pass across the membrane, enters the cavity. D) Example for a lipid intrusion event. While the fluorescent signal for the lipid dye (violet) increases due to the membrane invading the cavity space, the translocation solute is pushed out of the cavity (red), leading to a decrease in fluorescent signal. The nanopore is blocked by the lipid bilayer, preventing the passage of the control substrate (green). E) During membrane rupture, the enclosed transport solute rapidly evades from the cavity (red), while the control solute diffuses in (green). This figure was reused with permission from 21. Please click here to view a larger version of this figure.
The technique presented here allows a highly parallel analysis of membrane protein transport. Reconstituted membrane protein systems can directly be applied to the biochip, making the adaption of theoretically every membrane transporter or channel possible. Transport analysis is only limited by the establishment of a fluorescent read-out system, either via direct fluorescence change (translocation of fluorophores or fluorescently labeled substrates) or indirect fluorescence change (pH-sensitive dyes, secondary enzymatic reactions). The latter, however have yet to be established.
Functional characterization of membrane channels or transporters on the single protein level is the main focus of this technique. In combination with an automated microscope, the establishment of screening applications for protein effectors is also possible. The assay setup allows the parallel recording of up to 8 chips, with multiple assay points on each chip. Because of complete separation of the chips, it is truly parallel, allowing for multiple experimental repeats including controls in a single experimental run, once the conditions have been established.
Critical steps during setting up an experiment are the preparation of truly unilamellar vesicles, the active reconstitution of the protein of choice, spreading efficiency of proteoliposomes on the chip surface and the retention capability of the suspended lipid bilayer toward the substrates used in the assay.
While the preparation of unilamellar vesicles is of basic importance for this assay to avoid the establishment of multilamellar membrane sheets on the chip surface during suspended lipid bilayer formation, it is rather easy to ensure unilamellarity of the vesicles via sonicating the liposomes during preparation.
The direct application of proteoliposomes is the big advancement of the method presented. In contrast to previous approaches more complex and medically relevant proteins can now be targeted, requiring of course the prior establishment of a reconstitution protocol yielding functional protein of choice in LUVs. Additionally, membrane proteins possessing large extra-membrane domains decrease the formation of proper SSMs. This has to be optimized for each new protein, for example by varying the CaCl2 concentration.
The reconstitution ratio of the protein of interest has to be chosen carefully. The analysis of single protein events is desired and accomplishable using the here presented technique. The amount of reconstituted protein should therefore not exceed 1-2 functional protein units per pore-spanning membrane patch assuming an ideal reconstitution and that the membrane protein is stochastically distributed across the bilayer. Additionally the directionality of the protein after reconstitution should also be kept in mind, as it has not to be equal between both directionalities.
Finally once all these issues are carefully checked and optimized, none of the used substrates may display any unspecific membrane permeability of the pore-spanning membrane. Positively charged fluorophores, like most rhodamine-based dyes in the red spectral regime tend to quickly overcome the membrane barrier. Strong hydrophobic interactions can also lead to an unspecific attachment to the lipid bilayer. Both properties are not desired.
The study of ion channels in a qualitative manner is also imaginable. Screening for channel effectors using a binary Yes/No readout could be thought of using ion-sensitive dyes. The biggest obstacle here would be the establishment of an at least partly ion-retaining lipid bilayer. This might only be achievable via surface modification of the nanopore chip.
The authors have nothing to disclose.
We thank Barbara Windschiegl for her help in establishing SOPs; Dennis Remme for his work on the NanoCalcFX software and Alina Kollmannsperger, Markus Braner and Milan Gerovac for helpful suggestions on the manuscript. The German-Israeli Project Cooperation (DIP) provided by the DFG and the Federal Ministry of Education and Research to R.T., as well as the Federal Ministry of Economics and Technology (ZIM R&D Project) to R.T. and Nanospot GmbH supported this work.
Reagent | |||
[2-(Trimethylammonium)ethyl] methanethiosulfonate |
Toronto Research Chemicals Inc. | T792900 | MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 °C. Use immediately (30 minutes) after solubilization in buffer. |
1 ml gas-tight syringe | Hamilton | #1001 | |
10 ml round flask | Schott Duran | ||
2.7 mm glas beads | Roth | N032.1 | |
2-Propanole | Roth | 9866.5 | |
30 cm Luer-Lock Extension Tube | Sarstedt | 744304 | |
Acetone | Roth | 5025.5 | |
Bio-Beads SM-2 Adsorbent | Bio-Rad | 152-3920 | need to be activated before first use |
CaCl2 Dihydrat | Roth | HN04.3 | |
Calcein | Sigma | C0875 | store dark at -20 °C |
Chloroform reagent grade | VWR Chemicals | 22711324 | |
DOPEATTO390 | ATTO-TEC | AD 390-165 | store dark at -20 °C |
Ethanol absolute | Sigma-Aldrich | 32205 | |
Injekt Single-use syringe | Braun | 460 60 51V | |
Injekt-F single-use syringe | Braun | 91 66 017V | |
Keck clips | Schott | KC29 | |
L-α-Phosphatidylcholine, 20% (Soy) | Avanti Polar Lipids | 5416016 | store under inert gas at -20 °C |
NaCl 99.5% p.a. | Roth | 3957.2 | |
Nanopore E100 wafer/chips | Micromotive (Mainz/Germany) | available on request | |
Nucleopore Track-Etch Membrane 0.4 µm | Whatman | 800282 | |
Oregon Green Dextran 488 (70 kDa) | life Technologies | D-7173 | store dark at -20 °C |
Oy647 | Luminartis (Münster/Germany) | OY-647-T-1mg | store dark at -20 °C |
Rotilabo-syringe filtration, unsterile, pore-size 0.22 µm | Roth | P 818.1 | |
Sephadex G-50 | Sigma-Aldrich | G5080 | column material for size exclusion chromatography |
Silastic MDX4-4210 | Dow Corning | curing agent for chip fixation onto cover glass support | |
sticky-Slide 8-well | ibidi | 80828 | multi-well chamber for the mounting onto glass slides (chip holder) |
Three-way stopcock blue | Sarstedt | 744410001 | |
Tris Pufferan 99.9% Ultra Quality | Roth | 5429.2 | |
Triton-X 100 | Roth | 6683.1 | |
Whatman 0.2 µm cellulose nitrate membrane filter | Roth | NH69.1 | |
Name | Company | Catalog Number | Comments |
Equipment | |||
Büchi 461 water bath | Büchi | ||
Büchi Rotavapor RE 111 | Büchi | ||
Cary Eclipse Fluorescence Spectrophtometer | Varian | ||
LiposoFast Mini Extruder | Avestin | ||
Membrane pump | Vaccubrand | 15430 | |
Nanosight Nanoparticle Tracking Microscope | Malvern / Nanosight | LM 14C | |
NyONE microscope | Synentec | available on request | |
Pump control | Vaccubrand | CVC 2II | |
Sonicator bath Sonorex RK100H | Brandelin electronic | 31200001107477 | |
Vaccum pump RC5 | Vaccubrand | 1805400204 | |
Water bath W13 | Haake | 002-9910 | |
Plasma Cleaner PDC-37G | Harrick Plasma | PDC-37G | |
Name | Company | Catalog Number | Comments |
Software | |||
ImageJ | Open Source | http://imagej.nih.gov/ij/ | scientific image processing software |
NanoCalcFX | Freeware | http://sourceforge.net/projects/nanocalc/ | data analysis/evaluation software for massive transport kinetic datasets |
NTA 2.3 Analytical Software | Nanosight | data acquisition and analysis software for nanoparticle tracking microscope | |
NTA 2.3 Temperature Comms | Nanosight | temperature controle software for nanoparticle tracking microscope |