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Cell-free synthesis of a functional membrane transporter into a tethered bilayer lipid membrane Julius L. Zieleniecki, Yagnesh Nagarajan, Shane Waters, Jay Rongala, Vanessa C Thompson, Maria Hrmova, and Ingo Köper Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04059 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016
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Cell-free synthesis of a functional membrane transporter into a tethered bilayer lipid membrane Julius L. Zieleniecki1, Yagnesh Nagarajan2, Shane Waters2, Jay Rongala2, Vanessa Thompson1, Maria Hrmova2,*, Ingo Köper1,* 1) Flinders Centre for Nanoscale Science and Technology School of Chemical and Physical Sciences Flinders University, Bedford Park, South Australia 5042, Australia 2) Australian Centre for Plant Functional Genomics School of Agriculture, Food, and Wine University of Adelaide, Glen Osmond, South Australia 5064, Australia Keywords: anion efflux transporter, electrochemical impedance spectroscopy, transport function, model membranes.
ABSTRACT: Eukaryotic cell-free synthesis to incorporate the large and complex multi-span plant membrane transporter Bot1 in a functional form into a tethered bilayer lipid membrane. The electrical properties of the protein-functionalized tethered bilayer were measured using electrochemical impedance spectroscopy, and revealed a pH-dependent transport of borate ions through the protein. The efficacy of the protein synthesis has been evaluated using immunoblot analysis.
Introduction Membrane proteins are essential components of every living cell. They mediate the transport of solutes across cell membranes, are responsible for cell-cell interactions, detect external stimuli, and facilitate many other critical cellular functions. Membrane protein malfunction causes a wide variety of diseases and about 60% of pharmaceutical drugs target membrane proteins. However, membrane proteins often occur in low quantities in native tissues, and are difficult to produce, purify, and study under controlled conditions. The main challenge for studies of membrane proteins is to find a suitable host environment, i.e. a lipid membrane environment, allowing for correct protein function. Natural cell membranes have complex architectures consisting of a variety of constituents, including lipids, carbohydrates and proteins. This complexity makes systematic investigations of membrane-related processes difficult. To simplify the membrane architecture, model systems such as liposomes, lipid monolayers, nanodiscs or solid supported membranes are often used.1, 2, 3 Among the solid supported membranes, tethered bilayer lipid membranes (tBLMs) offer a useful platform by providing a robust and electrochemically insulating bilayer membrane.4 In contrast to free-standing bilayer (or black) lipid membranes, tBLMs offer a very stable system, allowing to perform experiments over extended time periods and give access to a wide range of surface analytical techniques.4, 5, 6, 7 A tBLM consists of a lipid bilayer, where the proximal leaflet is covalently bound to a solid support via a short spacer group (Figure 1).7, 8, 9, 10 Attachment to a surface allows for the use of a variety of surface analytical techniques, such as ACS Paragon Plus Environment
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atomic force microscopy or electrochemical impedance spectroscopy (EIS).7, 11, 12 Membrane proteins incorporated in lipid bilayers that are directly supported on hydrophilic substrates suffer from non-physiological interactions with the solid support leading to a loss of protein dynamics and function.13 tBLMs circumvent this problem by lifting the membrane off the surface and providing an additional space, which allows for proper protein flexibility.14 Anchoring the bilayer onto the surface yields a stable system, which is suitable for systematic investigations of the structure and function of the membrane architecture and embedded proteins. The spacer between the support and the lipid bilayer provides room to accommodate the extramembrane components of membrane-embedded proteins, and serves as a reservoir for transported ions and small molecules. By using a conducting surface, the transport properties of proteins can be characterized electrochemically.7, 12, 15, 16 There are two common techniques for the incorporation of membrane proteins into a preformed bilayer. After a membrane protein is purified, stabilized, and solubilized in aqueous media, it can be added directly to a preformed membrane.7 However, this method is limited to rather simple protein structures, and often it is difficult to preserve protein function after incorporation. A second approach is to synthesize a membrane protein in vesicles and use these proteo-liposomes to form a lipid bilayer.4 Successful examples using this approach have been documented, although in most cases they are limited to less complex protein structures.4 A third and relatively new alternative is co-translational insertion of a membrane protein directly into a pre-formed membrane, for example into a tBLM, using a cell-free synthesis approach.17, 18, 19, 20 A wheat germ-based cell-free synthesis system enables rapid production of proteins and is a versatile method to synthesize complex membrane proteins.18, 19 This open system allows for supplementation of any components, such as ligands or enzymes, at any time during the cell-free synthesis reaction. The technique was originally developed to study transcription and translation processes, and it is currently used in a variety of studies stretching from basic research to large scale industrial experiments.17 The rapid advancements in cell-free synthesis enable more cross-disciplinary research into the structure and functions of membrane proteins. Cell-free synthesis of membrane proteins has been applied primarily using vesicles instead of solid supported membranes.21, 22 However, in vesicles it is difficult to test the function of membrane proteins, especially ion channel proteins. Yet, ion channel proteins represent a major class of membrane proteins involved in almost all cellular processes. So far few reports of cell-free membrane protein synthesis in supported membranes directly demonstrated protein function, and no functional proof of ion transport has been shown.23, 24, 25 Here, eukaryotic cell-free protein synthesis to embed a plant membrane transport protein, designated Bot1, into a tBLM. (Figure 1) Bot1 shares high homology with anion transporters and is one of several proteins known to mediate tolerance of barley plants to high soil borate [B(OH)4]- concentrations.26, 27 Borate anion efflux is pH-dependent, with a loss of function at pH values approaching the pKa of boric acid, (9.24).27 Recently, the exact mechanism of borate efflux by Bot1 was described revealing that it occurs through the quantum tunneling process.27 In barley roots, the efflux of borate ions can be reduced by anion channel inhibitors, although this causes metabolic inhibition. The successful incorporation of the Bot1 transport protein into the tBLM was shown by immunoblot analysis, whereas the transport properties of Bot1 were monitored by EIS. Due to very low currents involved and subsequent requirements for highly insulating membranes, the monitoring of ion currents through anion membrane transport proteins is challenging.4 How-
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ever, this approach is also more sensitive than commonly employed optical detection techniques used to assess protein function.
Figure 1. Schematic view of a tBLM containing the transmembrane transporter Bot1. The system is formed by first forming a DPhyTL monolayer by self-assembly on a gold substrate. Fusion of this layer with DPhyPC vesicles leads to the formation of a bilayer and the protein is incorporated into the membrane via eukaryotic cell-free synthesis. The funnel-like structure (in cyan) represents Bot1, whereby its cytoplasmic α-helices26 point to the distal side of the bilayer. Materials and Methods Materials The anchor lipid DPhyTL (2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-d,L-α-lipoic acid ester) was synthesized as described previously.28 DPhyPC (1,2Diphytanoylphosphatidylcholine) was obtained from Avanti Polar Lipids (Alabaster, AL). HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), NaCl and BTP (bis(tris(hydroxymethyl) methylamino)-propane) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Membrane formation DPhyTL monolayers were self-assembled as described elsewhere.7 Samples were rinsed with ethanol and dried under a stream of nitrogen. Monolayers were completed to bilayers by fusion with small unilamellar DPhyPC vesicles (50 nm, generated by extrusion, 2 mg lipid per mL in 1 mM NaCl). Cell-free synthesis of Bot1 The cDNA of Bot1 was amplified from the Sahara cultivar,20 and cloned into the cell-free pEU-EO1 (frame N2) expression vector (CellFree Sciences, Tsurumi-ku, Yokohama, Japan) containing an N-terminal 6xHis tag and a Tobacco-Etch Virus proteolytic site. The introduced tags did not change the function of the protein.27 Wheat-germ cell-free synthesis of Bot1 was performed in a de-coupled mode as described previously.18,27 Briefly, a transcription reaction was set up and purified mRNA, along with the wheat germ extract, other cofactors and amino acids were added to a preformed tBLM. The protein synthesis was carried out for 24 h at room temperature without shaking.
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Electrochemical Impedance Spectroscopy Measurements were conducted using an Autolab PGSTAT 30 impedance spectrometer as described before.7 Spectra were recorded for frequencies between 0.5 mHz and 1 MHz at a 0 V bias potential and with a 10 mV AC modulation amplitude. Raw data were analyzed using the ZVIEW software package (version 3.3B Scribner Associates). Data was fitted to an equivalent circuit of capacitors and resistors, Rel(RmemCmem)Csp.4 Final values were normalized to the electrode surface area (0.28 cm2). To exploit the sodium-dependent transport of borate, EIS measurements were performed in HEPES-BTP buffer (20 mM HEPES) or in HEPES-BTP buffer containing borate (20 mM HEPES, 50 mM boric acid), at various pH values and in the presence or absence of 5 mM NaCl. Immunoblot Analysis Once transport measurements were completed, the membrane was dissolved in 15 µl of 2% (w/v) SDS buffer (50 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1% (v/v) β-mercaptoethanol, 12.5 mM EDTA, 0.02% (w/v) bromophenol blue). Immunoblot analysis was performed with a crude serum of Bot1 raised in rabbits using the CSVDKDLKSLKDAVLREGDE immunogenic peptide of Bot1.27 Immunoblot profiles were detected using the Novex® ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Carlsbad, CA, USA) or with the BCIP/NBT-purple liquid reagent (Sigma-Aldrich) according to manufacturer’s protocols. Results and Discussion Tethered bilayer membranes consisting of a proximal DPhyTL and a distal DPhyPC outer leaflet were formed by self-assembly and vesicle fusion following established protocols.7 The successful formation of an insulating bilayer was monitored by EIS. In an EIS experiment, the system is probed by applying an AC signal between working and counter electrodes over a wide frequency range. The resulting current is analyzed and can be plotted in Bode plots (Figure 2). In this representation, the magnitude of the impedance and the phase shift are plotted as a function of frequency. To extract electrical parameters, the data can be fitted to an equivalent circuit of resistive and capacitive elements (Table 1). Resistive elements can be identified by a low-phase angle and a constant impedance, whereas capacitive elements result in a high phase angle and a -1-slope in the impedance. Here, the lipid bilayer has been described by a resistor and a capacitor in parallel, in series with an additional capacitor, representing the gold interface and spacer region (inset in Figure 2a). The results presented represent a typical experiment. The experiment has been performed successfully multiple times, however the electrical properties of the membrane typically vary between experiments, which makes the calculation of averages somewhat challenging.4 The formation of a bilayer led to an increase in membrane resistance and a decrease in capacitance, in good agreement with previously published findings.7 The bare bilayer showed little sensitivity towards the presence of borate ions. The electrical properties in HEPES buffer with and without borate were almost identical (data not shown). The Bot1 transporter is a large membrane protein with a complex architecture and similar to its homologues, contains membrane and cytoplasmic components.29 The atomistic structure of Bot1 has recently been reported, and the study reveals its complex architecture consisting of membrane and cytoplasmic α-helices.27 The tBLM was exposed to a wheat germ-based
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cell-free synthesis reaction system containing Bot1 mRNA, which led to the synthesis of the functional protein and its simultaneous co-translational incorporation into the membrane. The barley Bot1 ion transporter is classified within the 2.A.31 family of anion exchangers; permeation properties of some family members were reported to be sodium dependent.30 Functional incorporation of the Bot1 protein was monitored using EIS to measure the electrical properties of the bilayer membrane. EIS does not resolve single ion channel translocations, as for example those that can be measured in patch-clamp experiments, but rather yields the average electrical properties of the membrane system. Transport of ions across the bilayer membrane can be detected by changes in the membrane resistance, while the membrane capacitance is an indicator of the membrane integrity.31 Cell-free synthesis led to a decrease in membrane resistance (Table 1, row 1), however the electrical values indicated that a well-sealing bilayer was conserved. After Bot1 incorporation (Figure 2), the electrical properties of the protein-functionalized tBLM were measured as a function of pH and in the presence and absence of sodium ions (5 mM NaCl).
Figure 2. Bode plots of a tBLM containing cell-free synthesized Bot1. Symbols represent data points, whereas solid lines are fits to the equivalent circuit shown in the inset in (a); squares ( , ) represent data in HEPES-BTP buffer only, stars (★,) the data in HEPES-BTP buffer with 5 mM boric acid. a) pH 6, no NaCl, b) pH 6, 5 mM NaCl, c) pH 7.5, 5 mM NaCl, d) pH 9, 5 mM NaCl. At pH 6, boric acid exists predominantly in a non-dissociated form. Under these conditions, the membrane resistance and capacitance showed very little boric acid dependence. The membrane resistance even increased slightly upon addition of boric acid. (Figure 2a) Similarly, the addition of sodium ions at pH 6 did not yield a measurable change in the electrical properties of the membrane, indicating that there was no transport through the Bot1 protein, since at this pH there were no borate ions present that could be transported. (Figure 2b) The resistance and capacitance values of the membrane are determined by the transport of the electrolyte molecules though the membranes and potential membrane defects. Values in the MΩ cm2 (resistance) and µF cm-2 (capacitance) ranges indicate a well-sealed membrane, in a good agreement with literature (Table 1). The chosen concentration of sodium was based on previous patch-clamp experiments, where 5 mM NaCl showed a significant increase in conACS Paragon Plus Environment
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ductivity. The results agree with patch-clamp data of Bot1 co-translationally incorporated in giant liposomes and with the Xenopus oocyte data.32 At pH 7.5, the boric acid concentration of 5 mM corresponds to a borate concentration of 90 µM. The addition of boric acid to the membrane led to a 50% decrease in membrane resistance from 3.19 MΩcm2 to 1.49 MΩcm2 (Table 1, rows 6 and 7), demonstrating the transport of borate ions across the membrane. (Figure 2c) A constant membrane capacitance showed that the membrane integrity remained unchanged (Table 1). At pH 9, the borate concentration is even higher (1.8 mM). However, the observed effect, i.e. the difference in membrane resistance in the presence and absence of borate anions, was only slightly higher than at pH 7.5 (Table 1, rows 8 and 9). The resistance in the presence of borate decreased by about 60% (Figure 2d). This suggests that the maximal borate transport rate has been reached, which is in agreement with established limits of borate transport in vivo, near pH 9.24,27, 33 and is in good agreement with recent in vitro and in vivo experiments using similar conditions.32 The high pH could also destabilize or partially unfold the Bot1 protein structure. The unfolded protein would lead to a significant decrease or abolishment of the transport function of Bot1. The high pH had a direct, but small effect on the membrane itself, as seen by the decrease of the overall resistance. However, the membrane capacitance remained relatively constant, indicating that there was no major change in the membrane integrity. EIS does not inform about the orientation of the incorporated protein. However, the protein structure would suggest, that the large extra-membrane part is located on the distal side of the bilayer.32 (Figure 1) Due to the molecular structure or the tether lipids, the space between the bilayer and the support is rather limited, however it has been shown that although fully tethered tBLMs can accommodate rather large protein components.4 To verify the presence of the synthesized protein, immunoblot analysis was performed. After the electrochemical measurements were completed, the lipids and proteins were stripped off the gold surface with 2% (w/v) SDS and the resultant mixture was analyzed. (Figure 3) The presence of the Bot1 protein band of a molecular mass of approximately 65 kDa (monomeric form; Figure 3, lane 2) and its trimeric assembly (arrows) were clearly visible. Variation in the mobility of Bot1 forms synthesized through cell-free synthesis may be explained by differential solvation of proteins by SDS.34 The protein bands were not observed in a control sample lacking Bot1 mRNA (lane 1) confirming the successful Bot1 synthesis and incorporation into the tBLM using wheat-germ cell free synthesis.
Table 1. Electrical properties of the tBLM with cell-free synthesized Bot1. Experimental data have been fitted to an equivalent circuit (Figure 2), and normalized to the electrode area. Data is from a representative experiment, errors are deviations derived from the fitting procedure.
Before cell-free synthesis, pH 6.0 pH 6.0, no NaCl, no borate pH 6.0, no NaCl, borate
Rmem/MΩ cm2 14.0 ± 0.3
Cmem/ µF cm-2 1.11 ± 0.08
Csp/µF cm-2 2.11 ± 0.09
3.58 ± 0.21
1.18 ± 0.03
2.81 ± 0.08
4.68 ± 0.24
1.24 ± 0.03
2.46 ± 0.06
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3.84 ± 0.22
1.23 ± 0.03
2.49 ± 0.07
3.64 ± 0.31 3.19 ± 0.19
1.24 ± 0.04 1.18 ± 0.03
2.59 ± 0.11 2.86 ± 0.09
1.49 ± 0.10
1.17 ± 0.03
2.86 ± 0.09
1.76 ± 0.12
1.11 ± 0.03
3.57 ± 0.13
0.73 ± 0.044
1.07 ± 0.03
3.93 ± 0.13
Figure 3. Immunoblot analysis of Bot1 synthesized via wheat-germ cell-free synthesis in a decoupled mode in an electro-impedance spectroscopy cell. Lanes 1 and 2 represent translation reactions with no mRNA and with Bot1 mRNA being added, respectively. Arrows represent monomeric and trimeric Bot1. Immunoblot was developed with a rabbit antibody raised against Bot1 and a secondary antibody conjugated to a horse-radish peroxidase. The blots were detected using a chemiluminescent substrate. Standards in kDa indicate molecular masses. Results shown are for a representative experiment; these data have been reproduced at least ten times. Conclusion Bot1, a large plant membrane transporter of a complex structure was inserted into a tBLM using eukaryotic cell-free synthesis in a de-coupled mode. Functional properties of Bot1 were determined by monitoring the pH-dependent transport of borate anions using EIS. This ap-
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proach offers the possibility to study more complex membrane proteins in environments that mimics natural membranes, preserves the function of membrane proteins and allows to use of a wide variety of surface analytical techniques. In the current work, EIS has been used to probe functional properties of the Bot1 protein, but the cell-free synthesis approach into a tBLM allows for many other complementary techniques to be employed in the future.
AUTHOR INFORMATION Corresponding Author [email protected] [email protected]. Funding Sources No competing financial interests have been declared. ACKNOWLEDGMENT This work was partially funded by the Australian Research Council grant DP120100900.
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27. Nagarajan, Y.; Rongala, J.; Luang, S.; Singh, A.; Shadiac, N.; Hayes, J.; Sutton, T.; Gilliham, M.; Tyerman, S.; McPhee, G.; Voelcker, N. H.; Mertens, H. D. T.; Kirby, N.; Lee, J.G.; Yingling, Y. G.; Hrmova, M. Na+-Dependent Anion Transport by a Barley Efflux Protein Revealed through an Integrative Platform. The Plant Cell 2015. 28. Atanasov, V.; Atanasova, P. P.; Vockenroth, I. K.; Knorr, N.; Koper, I. A molecular toolkit for highly insulating tethered bilayer lipid membranes on various substrates. Bioconjugate Chemistry 2006, 17 (3), 631-637. 29. Schnurbusch, T.; Hayes, J.; Hrmova, M.; Baumann, U.; Ramesh, S. A.; Tyerman, S. D.; Langridge, P.; Sutton, T. Boron Toxicity Tolerance in Barley through Reduced Expression of the Multifunctional Aquaporin HvNIP2;1. Plant Physiology 2010, 153 (4), 1706-1715. 30. Saier, M. H.; Reddy, V. S.; Tamang, D. G.; Västermark, Å. The Transporter Classification Database. Nucleic Acids Research 2014, 42 (D1), D251-D258. 31. Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Impedance analysis of supported lipid bilayer membranes: a scutiny of different preparation techniques. Biochimica et Biophysica Acta 1996, 1279, 169-180. 32. Hrmova, M.; Nagarajan, Y.; Rongala, J.; Luang, S.; Singh, A.; Shadiac, N.; Hayes, J.; Sutton, T.; Gilliham, M.; Tyerman, S.; McPhee, G.; Voelcker, N. H.; Mertens, H. D. T.; Kirby, N.; Lee, J.-G.; Yingling, Y. G. Na+-Dependent Anion Transport by a Barley Efflux Protein Revealed through an Integrative Platform. The Plant Cell 2015. 33. Hayes, J. E.; Reid, R. J. Boron Tolerance in Barley Is Mediated by Efflux of Boron from the Roots. Plant Physiol. 2004, 136 (2), 3376-3382. 34. Rath, A.; Glibowicka, M.; Nadeau, V. G.; Chen, G.; Deber, C. M. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (6), 1760-1765.
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Article
Cell-free synthesis of a functional membrane transporter into a tethered bilayer lipid membrane Julius L. Zieleniecki, Yagnesh Nagarajan, Shane Waters, Jay Rongala, Vanessa C Thompson, Maria Hrmova, and Ingo Köper Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04059 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016
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Cell-free synthesis of a functional membrane transporter into a tethered bilayer lipid membrane Julius L. Zieleniecki1, Yagnesh Nagarajan2, Shane Waters2, Jay Rongala2, Vanessa Thompson1, Maria Hrmova2,*, Ingo Köper1,* 1) Flinders Centre for Nanoscale Science and Technology School of Chemical and Physical Sciences Flinders University, Bedford Park, South Australia 5042, Australia 2) Australian Centre for Plant Functional Genomics School of Agriculture, Food, and Wine University of Adelaide, Glen Osmond, South Australia 5064, Australia Keywords: anion efflux transporter, electrochemical impedance spectroscopy, transport function, model membranes.
ABSTRACT: Eukaryotic cell-free synthesis to incorporate the large and complex multi-span plant membrane transporter Bot1 in a functional form into a tethered bilayer lipid membrane. The electrical properties of the protein-functionalized tethered bilayer were measured using electrochemical impedance spectroscopy, and revealed a pH-dependent transport of borate ions through the protein. The efficacy of the protein synthesis has been evaluated using immunoblot analysis.
Introduction Membrane proteins are essential components of every living cell. They mediate the transport of solutes across cell membranes, are responsible for cell-cell interactions, detect external stimuli, and facilitate many other critical cellular functions. Membrane protein malfunction causes a wide variety of diseases and about 60% of pharmaceutical drugs target membrane proteins. However, membrane proteins often occur in low quantities in native tissues, and are difficult to produce, purify, and study under controlled conditions. The main challenge for studies of membrane proteins is to find a suitable host environment, i.e. a lipid membrane environment, allowing for correct protein function. Natural cell membranes have complex architectures consisting of a variety of constituents, including lipids, carbohydrates and proteins. This complexity makes systematic investigations of membrane-related processes difficult. To simplify the membrane architecture, model systems such as liposomes, lipid monolayers, nanodiscs or solid supported membranes are often used.1, 2, 3 Among the solid supported membranes, tethered bilayer lipid membranes (tBLMs) offer a useful platform by providing a robust and electrochemically insulating bilayer membrane.4 In contrast to free-standing bilayer (or black) lipid membranes, tBLMs offer a very stable system, allowing to perform experiments over extended time periods and give access to a wide range of surface analytical techniques.4, 5, 6, 7 A tBLM consists of a lipid bilayer, where the proximal leaflet is covalently bound to a solid support via a short spacer group (Figure 1).7, 8, 9, 10 Attachment to a surface allows for the use of a variety of surface analytical techniques, such as ACS Paragon Plus Environment
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atomic force microscopy or electrochemical impedance spectroscopy (EIS).7, 11, 12 Membrane proteins incorporated in lipid bilayers that are directly supported on hydrophilic substrates suffer from non-physiological interactions with the solid support leading to a loss of protein dynamics and function.13 tBLMs circumvent this problem by lifting the membrane off the surface and providing an additional space, which allows for proper protein flexibility.14 Anchoring the bilayer onto the surface yields a stable system, which is suitable for systematic investigations of the structure and function of the membrane architecture and embedded proteins. The spacer between the support and the lipid bilayer provides room to accommodate the extramembrane components of membrane-embedded proteins, and serves as a reservoir for transported ions and small molecules. By using a conducting surface, the transport properties of proteins can be characterized electrochemically.7, 12, 15, 16 There are two common techniques for the incorporation of membrane proteins into a preformed bilayer. After a membrane protein is purified, stabilized, and solubilized in aqueous media, it can be added directly to a preformed membrane.7 However, this method is limited to rather simple protein structures, and often it is difficult to preserve protein function after incorporation. A second approach is to synthesize a membrane protein in vesicles and use these proteo-liposomes to form a lipid bilayer.4 Successful examples using this approach have been documented, although in most cases they are limited to less complex protein structures.4 A third and relatively new alternative is co-translational insertion of a membrane protein directly into a pre-formed membrane, for example into a tBLM, using a cell-free synthesis approach.17, 18, 19, 20 A wheat germ-based cell-free synthesis system enables rapid production of proteins and is a versatile method to synthesize complex membrane proteins.18, 19 This open system allows for supplementation of any components, such as ligands or enzymes, at any time during the cell-free synthesis reaction. The technique was originally developed to study transcription and translation processes, and it is currently used in a variety of studies stretching from basic research to large scale industrial experiments.17 The rapid advancements in cell-free synthesis enable more cross-disciplinary research into the structure and functions of membrane proteins. Cell-free synthesis of membrane proteins has been applied primarily using vesicles instead of solid supported membranes.21, 22 However, in vesicles it is difficult to test the function of membrane proteins, especially ion channel proteins. Yet, ion channel proteins represent a major class of membrane proteins involved in almost all cellular processes. So far few reports of cell-free membrane protein synthesis in supported membranes directly demonstrated protein function, and no functional proof of ion transport has been shown.23, 24, 25 Here, eukaryotic cell-free protein synthesis to embed a plant membrane transport protein, designated Bot1, into a tBLM. (Figure 1) Bot1 shares high homology with anion transporters and is one of several proteins known to mediate tolerance of barley plants to high soil borate [B(OH)4]- concentrations.26, 27 Borate anion efflux is pH-dependent, with a loss of function at pH values approaching the pKa of boric acid, (9.24).27 Recently, the exact mechanism of borate efflux by Bot1 was described revealing that it occurs through the quantum tunneling process.27 In barley roots, the efflux of borate ions can be reduced by anion channel inhibitors, although this causes metabolic inhibition. The successful incorporation of the Bot1 transport protein into the tBLM was shown by immunoblot analysis, whereas the transport properties of Bot1 were monitored by EIS. Due to very low currents involved and subsequent requirements for highly insulating membranes, the monitoring of ion currents through anion membrane transport proteins is challenging.4 How-
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ever, this approach is also more sensitive than commonly employed optical detection techniques used to assess protein function.
Figure 1. Schematic view of a tBLM containing the transmembrane transporter Bot1. The system is formed by first forming a DPhyTL monolayer by self-assembly on a gold substrate. Fusion of this layer with DPhyPC vesicles leads to the formation of a bilayer and the protein is incorporated into the membrane via eukaryotic cell-free synthesis. The funnel-like structure (in cyan) represents Bot1, whereby its cytoplasmic α-helices26 point to the distal side of the bilayer. Materials and Methods Materials The anchor lipid DPhyTL (2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-d,L-α-lipoic acid ester) was synthesized as described previously.28 DPhyPC (1,2Diphytanoylphosphatidylcholine) was obtained from Avanti Polar Lipids (Alabaster, AL). HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), NaCl and BTP (bis(tris(hydroxymethyl) methylamino)-propane) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Membrane formation DPhyTL monolayers were self-assembled as described elsewhere.7 Samples were rinsed with ethanol and dried under a stream of nitrogen. Monolayers were completed to bilayers by fusion with small unilamellar DPhyPC vesicles (50 nm, generated by extrusion, 2 mg lipid per mL in 1 mM NaCl). Cell-free synthesis of Bot1 The cDNA of Bot1 was amplified from the Sahara cultivar,20 and cloned into the cell-free pEU-EO1 (frame N2) expression vector (CellFree Sciences, Tsurumi-ku, Yokohama, Japan) containing an N-terminal 6xHis tag and a Tobacco-Etch Virus proteolytic site. The introduced tags did not change the function of the protein.27 Wheat-germ cell-free synthesis of Bot1 was performed in a de-coupled mode as described previously.18,27 Briefly, a transcription reaction was set up and purified mRNA, along with the wheat germ extract, other cofactors and amino acids were added to a preformed tBLM. The protein synthesis was carried out for 24 h at room temperature without shaking.
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Electrochemical Impedance Spectroscopy Measurements were conducted using an Autolab PGSTAT 30 impedance spectrometer as described before.7 Spectra were recorded for frequencies between 0.5 mHz and 1 MHz at a 0 V bias potential and with a 10 mV AC modulation amplitude. Raw data were analyzed using the ZVIEW software package (version 3.3B Scribner Associates). Data was fitted to an equivalent circuit of capacitors and resistors, Rel(RmemCmem)Csp.4 Final values were normalized to the electrode surface area (0.28 cm2). To exploit the sodium-dependent transport of borate, EIS measurements were performed in HEPES-BTP buffer (20 mM HEPES) or in HEPES-BTP buffer containing borate (20 mM HEPES, 50 mM boric acid), at various pH values and in the presence or absence of 5 mM NaCl. Immunoblot Analysis Once transport measurements were completed, the membrane was dissolved in 15 µl of 2% (w/v) SDS buffer (50 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1% (v/v) β-mercaptoethanol, 12.5 mM EDTA, 0.02% (w/v) bromophenol blue). Immunoblot analysis was performed with a crude serum of Bot1 raised in rabbits using the CSVDKDLKSLKDAVLREGDE immunogenic peptide of Bot1.27 Immunoblot profiles were detected using the Novex® ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Carlsbad, CA, USA) or with the BCIP/NBT-purple liquid reagent (Sigma-Aldrich) according to manufacturer’s protocols. Results and Discussion Tethered bilayer membranes consisting of a proximal DPhyTL and a distal DPhyPC outer leaflet were formed by self-assembly and vesicle fusion following established protocols.7 The successful formation of an insulating bilayer was monitored by EIS. In an EIS experiment, the system is probed by applying an AC signal between working and counter electrodes over a wide frequency range. The resulting current is analyzed and can be plotted in Bode plots (Figure 2). In this representation, the magnitude of the impedance and the phase shift are plotted as a function of frequency. To extract electrical parameters, the data can be fitted to an equivalent circuit of resistive and capacitive elements (Table 1). Resistive elements can be identified by a low-phase angle and a constant impedance, whereas capacitive elements result in a high phase angle and a -1-slope in the impedance. Here, the lipid bilayer has been described by a resistor and a capacitor in parallel, in series with an additional capacitor, representing the gold interface and spacer region (inset in Figure 2a). The results presented represent a typical experiment. The experiment has been performed successfully multiple times, however the electrical properties of the membrane typically vary between experiments, which makes the calculation of averages somewhat challenging.4 The formation of a bilayer led to an increase in membrane resistance and a decrease in capacitance, in good agreement with previously published findings.7 The bare bilayer showed little sensitivity towards the presence of borate ions. The electrical properties in HEPES buffer with and without borate were almost identical (data not shown). The Bot1 transporter is a large membrane protein with a complex architecture and similar to its homologues, contains membrane and cytoplasmic components.29 The atomistic structure of Bot1 has recently been reported, and the study reveals its complex architecture consisting of membrane and cytoplasmic α-helices.27 The tBLM was exposed to a wheat germ-based
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cell-free synthesis reaction system containing Bot1 mRNA, which led to the synthesis of the functional protein and its simultaneous co-translational incorporation into the membrane. The barley Bot1 ion transporter is classified within the 2.A.31 family of anion exchangers; permeation properties of some family members were reported to be sodium dependent.30 Functional incorporation of the Bot1 protein was monitored using EIS to measure the electrical properties of the bilayer membrane. EIS does not resolve single ion channel translocations, as for example those that can be measured in patch-clamp experiments, but rather yields the average electrical properties of the membrane system. Transport of ions across the bilayer membrane can be detected by changes in the membrane resistance, while the membrane capacitance is an indicator of the membrane integrity.31 Cell-free synthesis led to a decrease in membrane resistance (Table 1, row 1), however the electrical values indicated that a well-sealing bilayer was conserved. After Bot1 incorporation (Figure 2), the electrical properties of the protein-functionalized tBLM were measured as a function of pH and in the presence and absence of sodium ions (5 mM NaCl).
Figure 2. Bode plots of a tBLM containing cell-free synthesized Bot1. Symbols represent data points, whereas solid lines are fits to the equivalent circuit shown in the inset in (a); squares ( , ) represent data in HEPES-BTP buffer only, stars (★,) the data in HEPES-BTP buffer with 5 mM boric acid. a) pH 6, no NaCl, b) pH 6, 5 mM NaCl, c) pH 7.5, 5 mM NaCl, d) pH 9, 5 mM NaCl. At pH 6, boric acid exists predominantly in a non-dissociated form. Under these conditions, the membrane resistance and capacitance showed very little boric acid dependence. The membrane resistance even increased slightly upon addition of boric acid. (Figure 2a) Similarly, the addition of sodium ions at pH 6 did not yield a measurable change in the electrical properties of the membrane, indicating that there was no transport through the Bot1 protein, since at this pH there were no borate ions present that could be transported. (Figure 2b) The resistance and capacitance values of the membrane are determined by the transport of the electrolyte molecules though the membranes and potential membrane defects. Values in the MΩ cm2 (resistance) and µF cm-2 (capacitance) ranges indicate a well-sealed membrane, in a good agreement with literature (Table 1). The chosen concentration of sodium was based on previous patch-clamp experiments, where 5 mM NaCl showed a significant increase in conACS Paragon Plus Environment
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ductivity. The results agree with patch-clamp data of Bot1 co-translationally incorporated in giant liposomes and with the Xenopus oocyte data.32 At pH 7.5, the boric acid concentration of 5 mM corresponds to a borate concentration of 90 µM. The addition of boric acid to the membrane led to a 50% decrease in membrane resistance from 3.19 MΩcm2 to 1.49 MΩcm2 (Table 1, rows 6 and 7), demonstrating the transport of borate ions across the membrane. (Figure 2c) A constant membrane capacitance showed that the membrane integrity remained unchanged (Table 1). At pH 9, the borate concentration is even higher (1.8 mM). However, the observed effect, i.e. the difference in membrane resistance in the presence and absence of borate anions, was only slightly higher than at pH 7.5 (Table 1, rows 8 and 9). The resistance in the presence of borate decreased by about 60% (Figure 2d). This suggests that the maximal borate transport rate has been reached, which is in agreement with established limits of borate transport in vivo, near pH 9.24,27, 33 and is in good agreement with recent in vitro and in vivo experiments using similar conditions.32 The high pH could also destabilize or partially unfold the Bot1 protein structure. The unfolded protein would lead to a significant decrease or abolishment of the transport function of Bot1. The high pH had a direct, but small effect on the membrane itself, as seen by the decrease of the overall resistance. However, the membrane capacitance remained relatively constant, indicating that there was no major change in the membrane integrity. EIS does not inform about the orientation of the incorporated protein. However, the protein structure would suggest, that the large extra-membrane part is located on the distal side of the bilayer.32 (Figure 1) Due to the molecular structure or the tether lipids, the space between the bilayer and the support is rather limited, however it has been shown that although fully tethered tBLMs can accommodate rather large protein components.4 To verify the presence of the synthesized protein, immunoblot analysis was performed. After the electrochemical measurements were completed, the lipids and proteins were stripped off the gold surface with 2% (w/v) SDS and the resultant mixture was analyzed. (Figure 3) The presence of the Bot1 protein band of a molecular mass of approximately 65 kDa (monomeric form; Figure 3, lane 2) and its trimeric assembly (arrows) were clearly visible. Variation in the mobility of Bot1 forms synthesized through cell-free synthesis may be explained by differential solvation of proteins by SDS.34 The protein bands were not observed in a control sample lacking Bot1 mRNA (lane 1) confirming the successful Bot1 synthesis and incorporation into the tBLM using wheat-germ cell free synthesis.
Table 1. Electrical properties of the tBLM with cell-free synthesized Bot1. Experimental data have been fitted to an equivalent circuit (Figure 2), and normalized to the electrode area. Data is from a representative experiment, errors are deviations derived from the fitting procedure.
Before cell-free synthesis, pH 6.0 pH 6.0, no NaCl, no borate pH 6.0, no NaCl, borate
Rmem/MΩ cm2 14.0 ± 0.3
Cmem/ µF cm-2 1.11 ± 0.08
Csp/µF cm-2 2.11 ± 0.09
3.58 ± 0.21
1.18 ± 0.03
2.81 ± 0.08
4.68 ± 0.24
1.24 ± 0.03
2.46 ± 0.06
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3.84 ± 0.22
1.23 ± 0.03
2.49 ± 0.07
3.64 ± 0.31 3.19 ± 0.19
1.24 ± 0.04 1.18 ± 0.03
2.59 ± 0.11 2.86 ± 0.09
1.49 ± 0.10
1.17 ± 0.03
2.86 ± 0.09
1.76 ± 0.12
1.11 ± 0.03
3.57 ± 0.13
0.73 ± 0.044
1.07 ± 0.03
3.93 ± 0.13
Figure 3. Immunoblot analysis of Bot1 synthesized via wheat-germ cell-free synthesis in a decoupled mode in an electro-impedance spectroscopy cell. Lanes 1 and 2 represent translation reactions with no mRNA and with Bot1 mRNA being added, respectively. Arrows represent monomeric and trimeric Bot1. Immunoblot was developed with a rabbit antibody raised against Bot1 and a secondary antibody conjugated to a horse-radish peroxidase. The blots were detected using a chemiluminescent substrate. Standards in kDa indicate molecular masses. Results shown are for a representative experiment; these data have been reproduced at least ten times. Conclusion Bot1, a large plant membrane transporter of a complex structure was inserted into a tBLM using eukaryotic cell-free synthesis in a de-coupled mode. Functional properties of Bot1 were determined by monitoring the pH-dependent transport of borate anions using EIS. This ap-
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proach offers the possibility to study more complex membrane proteins in environments that mimics natural membranes, preserves the function of membrane proteins and allows to use of a wide variety of surface analytical techniques. In the current work, EIS has been used to probe functional properties of the Bot1 protein, but the cell-free synthesis approach into a tBLM allows for many other complementary techniques to be employed in the future.
AUTHOR INFORMATION Corresponding Author [email protected] [email protected]. Funding Sources No competing financial interests have been declared. ACKNOWLEDGMENT This work was partially funded by the Australian Research Council grant DP120100900.
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27. Nagarajan, Y.; Rongala, J.; Luang, S.; Singh, A.; Shadiac, N.; Hayes, J.; Sutton, T.; Gilliham, M.; Tyerman, S.; McPhee, G.; Voelcker, N. H.; Mertens, H. D. T.; Kirby, N.; Lee, J.G.; Yingling, Y. G.; Hrmova, M. Na+-Dependent Anion Transport by a Barley Efflux Protein Revealed through an Integrative Platform. The Plant Cell 2015. 28. Atanasov, V.; Atanasova, P. P.; Vockenroth, I. K.; Knorr, N.; Koper, I. A molecular toolkit for highly insulating tethered bilayer lipid membranes on various substrates. Bioconjugate Chemistry 2006, 17 (3), 631-637. 29. Schnurbusch, T.; Hayes, J.; Hrmova, M.; Baumann, U.; Ramesh, S. A.; Tyerman, S. D.; Langridge, P.; Sutton, T. Boron Toxicity Tolerance in Barley through Reduced Expression of the Multifunctional Aquaporin HvNIP2;1. Plant Physiology 2010, 153 (4), 1706-1715. 30. Saier, M. H.; Reddy, V. S.; Tamang, D. G.; Västermark, Å. The Transporter Classification Database. Nucleic Acids Research 2014, 42 (D1), D251-D258. 31. Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Impedance analysis of supported lipid bilayer membranes: a scutiny of different preparation techniques. Biochimica et Biophysica Acta 1996, 1279, 169-180. 32. Hrmova, M.; Nagarajan, Y.; Rongala, J.; Luang, S.; Singh, A.; Shadiac, N.; Hayes, J.; Sutton, T.; Gilliham, M.; Tyerman, S.; McPhee, G.; Voelcker, N. H.; Mertens, H. D. T.; Kirby, N.; Lee, J.-G.; Yingling, Y. G. Na+-Dependent Anion Transport by a Barley Efflux Protein Revealed through an Integrative Platform. The Plant Cell 2015. 33. Hayes, J. E.; Reid, R. J. Boron Tolerance in Barley Is Mediated by Efflux of Boron from the Roots. Plant Physiol. 2004, 136 (2), 3376-3382. 34. Rath, A.; Glibowicka, M.; Nadeau, V. G.; Chen, G.; Deber, C. M. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (6), 1760-1765.
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Langmuir
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ACS Paragon Plus Environment
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Langmuir
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ACS Paragon Plus Environment
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