Concurrent In Vitro Synthesis and Functional Detection of Nascent

Department of Biotechnology, Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, Suita , Osaka 565-0871 ...
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Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX

Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential Masayuki Iwamoto,† Maie A. Elfaramawy,‡ Mariko Yamatake,† Tomoaki Matsuura,*,‡ and Shigetoshi Oiki*,† †

Department of Molecular Physiology and Biophysics, University of Fukui, Eiheiji-cho, Fukui 910-1193, Japan Department of Biotechnology, Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan

‡

S Supporting Information *

ABSTRACT: Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA potassium channel in lipid bilayers under an applied membrane potential. The channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional channels with the correct orientation. The singlechannel current of the first appearing nascent channel was captured, followed by the subsequent appearance of multiple channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane. KEYWORDS: in vitro transcription/translation, membrane protein, unassisted pathway, single channel current recording, membrane potential, droplet interface bilayer

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channel is formed as a homotetramer via the unassisted pathway.7 KcsA was synthesized using an E. coli-based reconstituted in vitro transcription/translation system (IVTT), where each component involved in the reaction was highly purified and reconstituted to carry out protein synthesis in a test tube (commercially available as PUREfrex1.0).11,12 In earlier studies, membrane proteins involving KcsA were synthesized in vitro in the presence of liposomes or nanodiscs, and the reconstituted membrane was served for transferring the synthesized proteins into a preformed lipid bilayer for functional measurements.13−16 However, in this study, channel synthesis and functional measurements are concurrent: the synthesis was performed in a water-in-oil droplet bounded by a membrane (droplet interface bilayer [DIB] method),17−20 where a membrane potential is applied electrophysiologically throughout protein synthesis (Figure 1B). Consequently, synthesized proteins are immediately incorporated into the membrane, and functional channels are readily detected at the single-channel level (concurrent expression and functional detection system). This system mimics the bacterial in vivo

embrane proteins undergo multiple steps after transcription and translation until they become functional in the cell membrane. Many membrane proteins are inserted into the membrane via the translocon-aided process,1−3 followed by folding and oligomerization, whereas some prokaryotic membrane proteins are spontaneously inserted into the membrane without the aid of the translocon machinery (unassisted pathway).4−6 In either pathway, the formation process of an active membrane protein involves partitioning to the membrane surface, insertion into the membrane, folding, and oligomerization (Figure 1A). However, there is a lack of information on the processes between the protein synthesis step and the formation of an active protein. Moreover, these four steps do not necessarily follow the same sequence of events as indicated by thermodynamic considerations.6 For example, synthesized proteins might be partially folded at the membrane interface before they are inserted into the membrane. Here, we established an in vitro membrane protein expression system specific for channel proteins, which serves as an analytical system to trace the functional formation of channel proteins at the single molecule level. The prokaryotic KcsA potassium channel was chosen because its structure and function have been studied extensively,7−10 and a functional © XXXX American Chemical Society

Received: December 15, 2017 Published: March 22, 2018 A

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Figure 1. KcsA channel expression system. (A) Channel protein expression using the reconstituted in vitro transcription/translation system (reconstituted IVTT; PUREfrex1.0) in the droplet interface bilayer (DIB). The synthesis of the KcsA potassium channel was performed in a droplet (left panel). The right panel shows a membrane system of the DIB such that the monolayer and bilayer coexist and were continuous to each other. The synthesized protein was partitioned to the interface of either the monolayer or bilayer, followed by insertion, folding, and tetramerization. (B) The concurrent expression and functional detection system. One microliter of IVTT solution was dropped into a drop-in-well chamber filled with azolectin-containing hexadecane. Two droplets were placed in contact spontaneously. Two glass microelectrodes were inserted into the droplets, and electrophysiological measurements were performed under a two-electrode voltage clamp configuration. Under the continuously applied membrane potential, the first-born channel was detected as a single-channel current. Inset shows a photo of the DIB system.

the adhesion of substances to the silver chloride (AgCl) electrode surfaces from the reconstituted IVTT and its products, which may introduce erroneous membrane potentials. The bilayer became unstable when a membrane potential was applied, presumably because of the condensed components in the reconstituted IVTT interfering with membrane integrity. Raising the azolectin concentration in hexadecane up to 100 mg/mL circumvented the vulnerability of the membrane, but the applied membrane potential was still limited to within ±100 mV. The membrane capacitance was ca. 400 pF, and with the specific capacitance of 0.8 μF/cm2,19 the bilayer area was ca. 50 000 μm2, resulting in relatively low electrical background noise. In this system, lipids for monolayers and bilayers were exclusively supplied from the oil phase. If lipids exist within the droplet as liposomes then the newly synthesized channel proteins cannot reach the surface monolayer or bilayer, but are trapped to the liposomes, which digresses the physiological process of cotranslational membrane interaction. Here, we used an open-channel mutant of the KcsA channel (H25R/E71A/E118A). The H25R/E118A double mutation allows the acid-activated KcsA channel to open at neutral pH (pH-independent mutation), and the E71A mutation allows the frequently inactivating KcsA to remain fully open (noninactivating mutation).21 Consequently, the pH-independent

expression system, which has a negative membrane potential, and differs from the eukaryotic counterpart which operates under a nearly null membrane potential. Under a defined membrane potential, this system allows for continuous monitoring of the formation of active channels, and we found, for the first time, nascent activities of first-born channels that appeared as single-channel currents. Lastly, we revealed that the membrane potential is not simply a technical convention for single-channel measurements, but is itself a factor for facilitating functional channel expression.

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RESULTS

Establishment of an Expression System Mimicking That of Bacteria. Two reconstituted IVTT-containing droplets (1 μL) were formed in a hexadecane oil phase, wherein azolectin was dispersed, and readily partitioned to the oil/water interface for monolayer formation (Figure 1B; see Methods). These droplets were spontaneously docked to form the bilayer in drop-in-wells. One of the droplets contains DNA encoding kcsA (5 nM), hereafter called the cis-droplet. Involvement of the IVTT in the trans-droplet aided in stabilizing the bilayer. A membrane potential was applied via a voltage-clamp circuitry using two glass microelectrodes inserted into the droplets. The glass microelectrodes minimize B

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology mutations allow for current recordings in the reconstituted IVTT-containing droplets, which operates exclusively at neutral pH. With the noninactivating E71A mutation,22,23 membraneincorporated channels can be immediately detected once they become functional. Thus, the appearance of channel currents denotes the presence of functional channels on the membrane, and the first appearance of nascent channel activity can be readily captured. DIB was formed and microelectrodes were inserted into the droplets at 25 °C, and protein synthesis was initiated by raising the temperature to the optimal 37 °C for IVTT (see Supplementary Figure S2 for distinct expression rates at different temperatures) by using a transparent heating plate below the chamber. Afterward, the membrane was voltageclamped throughout the experiment to detect the singlechannel currents of KcsA channels. This expression system mimics that found in a bacterial membrane, and we used this system to capture the first signal of the nascent KcsA channel after its synthesis. Concurrent Single-Channel Recordings During In Vitro Protein Synthesis. A representative current trace is shown in Figure 2 with an applied membrane potential of +50

Figure 3. Single-channel behavior of the nascent KcsA (H25R/ E118A/E71A) channel (A) compared to the in vivo-synthesized channel (B) at pH 7. The I−V curves (upper) and the single-channel current trace at +100 mV (lower) are shown. For the I−V curves, the error bars are shown for 4−10 observations.

osomes, and followed by bilayer reconstitution (hereafter referred to as the mature channel). The single-channel recording of the mature triple mutant channel at pH 7 is shown in Figure 3B.21 Compared to the mature channel, the single-channel conductance of the nascent channel was higher. This was probably due to the involvement of the E71A mutation in the triple mutant, which occasionally exhibits higher conductance (hereafter referred to as hyper),24 and the nascent channel mostly remains in the hyper state. Orientation of Expressed Channels. The orientation of the expressed channels was examined by exploiting the property of wild type (WT) channels exhibiting pH-sensitivity in channel gating; the WT channel becomes active when the pH on its cytoplasmic side is acidic. If the expressed channels are oriented with their cytoplasmic side facing the cis-droplet, as expected for the orientation of natively synthesized proteins, the channel current cannot be measured because of the cisdroplet’s neutral pH. In contrast, if the cytoplasmic side faces the trans-droplet, a current should be recorded as long as the trans-droplet pH is acidic. Here, the pH of either the cis- or trans-droplet was rendered acidic after channel synthesis. First, the WT channel was synthesized in a separated droplet, without forming the bilayer, at 37 °C (the pH of the cis- and transdroplets was 7.5) for 10 min. Then, the temperature was lowered to 25 °C to stop protein synthesis, and the pH of the cis- or trans-droplet was decreased by injecting an acidic solution (the final pH was 4.2; Figure 4A; see Methods). Finally, the bilayer was formed and a membrane potential (+50 or +75 mV) was applied at 25 °C. In the case of acidic cis-droplets, the macroscopic channel current appeared after a certain delay (∼1 min) post bilayer formation, and the membrane frequently broke immediately afterward (Figure 4B, black trace and inset). Conversely, in the case of acidic trans-droplets, no channel currents were observed, even after an hour post bilayer formation (Figure 4B, gray trace), indicating that oppositely oriented channels did not exist in the membrane. These observations indicated that the WT channel was inserted into the membrane with its cytoplasmic domain facing the cis-droplet, and the same is expected to be true for the triple mutant KcsA channel. The latter results without channel currents in the acidic trans-droplet for such a long time constitute a control experiment for this experimental system. No contaminant

Figure 2. Time course of the appearance of the KcsA channel. The IVTT reaction was started by raising the temperature to 37 °C at time 0 and immediately after, the membrane potential was set to +50 mV. In the time course presented, the first active channel emerged 28 min after the reaction was started, which was detected as a rectangular single-channel current (red arrow). Simultaneous emergence of multiple channels was observed in addition to one by one emergence as indicated in the inset.

mV. Single channel currents appeared spontaneously ∼28 min (the mean appearance time was 34.9 ± 2.57 min) after the initiation of channel synthesis (see Figure 6), and subsequently, the number of active channels increased (Figure 2). Big conductance steps were frequently observed (Figure 2 inset), in which the high-resolution recordings failed to detect stepwise increments within a short period of time. The simultaneous appearance of multiple active channels was likely in progress, and underlying events will be discussed later. Occasionally, a very large number of channels appeared and the membrane broke. Single-Channel Features of the Nascent Channel. The single-channel behavior of the nascent channel (i.e., the channel synthesized by the reconstituted IVTT) was examined. The single-channel current−voltage relationship exhibited a nearly linear relation in the ±100 mV range (Figure 3A). This behavior is compared with the same triple mutant channel synthesized in vivo from bacteria, which involves bacterial synthesis, solubilization, purification, incorporation into lipC

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Figure 4. Orientation of the membrane-inserted wild type KcsA channels. (A) The schematic procedure for the orientation analysis. The procedure exploits the feature of the wild type channel, exhibiting activation only when the pH of the cytoplasmic side become acidic. In this analysis, 5 nM of DNA encoding the wild type KcsA channel was included in the cis-droplet. First, the IVTT reaction was conducted for 10 min at 37 °C without the formation of the lipid bilayer. Next, the temperature was lowered to 25 °C and a small volume of concentrated acidic buffer (0.5 M, pH 3) was injected to the cis- or trans-droplet (the final pH was 4.2). After the formation of the lipid bilayer by docking the two droplets, the membrane potential was set to allow for observations of the emergence of the active channels in the lipid bilayer. (B) Typical time course of current recordings from the time of bilayer formation. cis acidic: black; trans acidic: gray. Active channels were observed only when the cis-droplet was acidic, indicating that the synthesized channels incorporated into the membrane with their cytoplasmic domain retained in the cis side. Inset shows the current appearance for the cis acidic condition after several tens of seconds after the bilayer formation.

Figure 5. Properties of KcsA channels synthesized using a reconstituted IVTT. (A) The amount of channel synthesis as a function of the DNA concentration. The IVTT was performed without the membrane but in the presence of [35S]-Methionine, and the total amount synthesized was quantified from autoradiography. (B) The time course of the channel synthesis in the presence of liposomes (large unilamellar vesicles [LUV]) and its localization. Lanes 1−10: IVTT solution in the presence of LUV was collected at the time indicated, and the supernatant fraction before (total) and after (sup) centrifugation were subjected for analysis. Lanes 11 and 12: LUV was added after 60 min of protein synthesis. Lanes 13 and 14: synthesis was performed for 60 min without LUV. (C) The first appearance time as a function of the DNA concentration. (D) Oligomeric state of the synthesized channel in the membrane. Protein synthesis was performed for 20 min in the presence of LUV and incubated for 30 min on ice. The supernatant fraction before (total) and after (sup) centrifugation were subjected to SDS-PAGE analysis without heat treatment. The in vivo synthesized sample was not heated before SDSPAGE. KcsA remains as a tetramer (right band). (E) Decomposition of the synthesis steps toward the formation of the functional channel. The procedure is schematically shown. The first appearance time was indistinguishable irrespective of whether the KcsA synthesis was performed in the presence (contacted) and absence (separated) of bilayer.

currents were observed, even though a huge amount of closed KcsA channels should exist in the membrane. Notably, the membrane remains stable as long as the channels are closed, suggesting that open channels may deform the membrane.25 Channel Synthesis, Localization and Oligomeric State. The properties and dynamics of the IVTT-synthesized KcsA channel was investigated. First, the amount of synthesized channels was quantified as a function of DNA concentration (Figure 5A). In this measurement, in vitro protein synthesis was performed in the absence of membranes, and the channel protein was solubilized with SDS for further autoradiography analysis (see Methods). The data indicate a linear increase in synthesized KcsA as a function of DNA concentration, saturating at over 500 pM of DNA.

Next, the localization of synthesized KcsA was examined. For this purpose, protein synthesis was performed in the presence of 13.8 mg/mL of azolectin liposomes (large unilamellar vesicles [LUV]) (see Methods), and the time course of synthesis was examined. Total protein synthesis and membrane fractions were evaluated to reveal the fraction of protein that integrated or bound to the lipid membrane (Figure 5B). Nearly 100% of the channels were found in the membrane fraction, even though the results do not specify whether the channels D

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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as monomers, and the efficacy of the formation of tetrameric functional channels was low. Nevertheless, once the functional channels appeared, they were in correct orientation with their cytoplasmic domain facing the cis-droplet. Acceleration of the First Appearance by the Membrane Potential. The membrane potential during synthesis was changed from +50 mV to +75 mV, and we found, surprisingly, that the first appearance time was substantially shortened from 34.9 ± 2.57 min to 15.0 ± 1.41 min (Figure 6).

were attached or inserted into the membrane (Figure 5B, lanes 1−10, and the plot below). On the other hand, almost all of the channels aggregated when protein synthesis was performed in the absence of liposomes (Figure 5B, lanes 13 and 14). Note that the membrane-associated fraction was proportional to the LUV concentration, which saturated at 13.8 mg/mL (Supplementary Figure S1). Furthermore, the addition of liposomes after protein synthesis did not prevent aggregation (Figure 5B, lanes 11 and 12). Thus, the presence of membranes rescues the synthesized channels from aggregation through a cotranslational interaction with the lipid membrane. In parallel, the first appearance time was also measured for different DNA concentrations. By increasing the DNA concentration in the droplet, the first appearance time was shortened only slightly (Figure 5C). A 100-fold reduction in DNA concentration (2.5 to 0.02 nM) prolonged the first appearance time only by an additional 10 min (ca. 2-fold), while the total amount of synthesized protein was reduced by at least 12-fold. These nonlinear characteristics suggest that the amount of synthesized channels was sufficient, and awaited functional channel formation by either remaining at the membrane interface or remaining nonactive in the membrane. The oligomeric state of the expressed channel in the membrane fraction was explored. The membrane fraction of KcsA was collected and electrophoresis was performed (Figure 5D). Protein synthesis was performed for 20 min in the presence of LUV, incubated for 30 min on ice, and then subjected to SDS-PAGE analysis without heat treatment. KcsA is known to form tetramers, even in the presence of 1% SDS, when not heated before SDS-PAGE. The results show that the synthesized channels remained predominantly as monomers, even after 30 min of incubation on ice. Finally, the maturation processes of the functional channels were decomposed into two stepsinterface partitioning and subsequent processesby the following method (Figure 1 and 5E). Similar to the previous experiments for channel orientation, channels were synthesized in an isolated droplet without bilayer formation at 37 °C. Channel synthesis was stopped by lowering the temperature to 25 °C, followed by bilayer formation, and then a membrane potential was applied. In this case, the channels undergo transcription/translation followed by partitioning to the monolayer interface, and the generation of functional channels proceeds once the bilayer is formed (Figure 1A). As a control experiment, the bilayer was formed from the beginning of channel synthesis without applying a membrane potential, and the rest of the procedures, such as the temperature control protocol, was identical. Channel synthesis was performed for 10 min, with and without the bilayer. Meanwhile, the channels were exclusively partitioned to the membrane interface in the isolated droplet, whereas part of the channels may be inserted into the bilayer in the control condition. When the temperature was lowered to stop protein synthesis, the amount of the synthesized channels was expected to be the same for both cases. The bilayer was formed for the isolated droplets, and a membrane potential was applied for both cases. Then, the first appearance time was measured beginning from the time the membrane potential was applied. Figure 5E shows that the first appearance time was indistinguishable between these two cases. This result suggests that the steps following partitioning, involving membrane insertion and oligomerization, are rate-limiting. In summary, the results indicate that the synthesized channels partition to the membrane and stay predominantly

Figure 6. First appearance time from the initiation of transcription/ translation as a function of the membrane potential. Number of the observation was 3−11.

Then, the membrane potential was set to negative. The first appearance time was 34.7 ± 3.94 min at −50 mV, and was substantially shortened (20.2 ± 2.48 min) as a more negative membrane potential (−75 mV) was applied. This was a unique voltage dependency, showing a nearly symmetrical bell shape. This symmetricity might possibly occur if the channel was inserted into the membrane in the opposite orientation when the membrane potential was kept negative. To examine this, a similar procedure to that of the previous channel orientation experiment was performed. Using the WT channel and setting the trans-droplet acidic, a negative membrane potential (−50 mV) was applied, but we failed to detect a channel current even after an hour (Supplementary Figure S3). This result indicates that the channels are inserted into the membrane with the correct orientation with either a positive or negative membrane potential. Applying the membrane potential was hitherto used to measure the channel current. Now that the experimental operation itself favors protein maturation (insertion/folding), the physiologically relevant membrane potential became a critical factor to be examined in the protein expression system of bacteria, which has a membrane potential of −200 mV, rather than the nearly null potential in eukaryotic endoplasmic reticulum membranes. The bell-shaped dependency provides clues for the underlying mechanism of expression. Chargedriven electrophoretic insertion is not likely because channels are correctly oriented in either polarity. Rather, the symmetry suggests a dependency on the magnitude of the electric field, and the physical features of membranes, such as bilayer thickness26 and tension,27 which are subject to change. These findings open a venue for further studies in the molecular mechanisms of protein−membrane interactions upon functional expression. E

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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DISCUSSION The present channel expression system mimics the in vivo bacterial membrane expression system via the unassisted pathway. In previous in vitro expression experiments, mass expression in the presence of membrane, but in the absence of membrane potential, was performed, and the membrane reconstituted channels were subsequently transferred to the lipid bilayer served for functional measurements.15,28 In contrast, our system allows for concurrent expression and functional detection; the transcribed and translated channels were immediately accessed to the membrane where a membrane potential was continually applied, and the firstborn channel activity was detected. This system generated the oriented-reconstitution of the KcsA channel, reproducing the in vivo system. Notably, capturing first-borne channels with first appearance times of nascent channel activity from initiation of protein synthesis is an unprecedented achievement of this study. Nascent channels had a higher conductance than WT channels, which is related to the E71A mutation24 introduced into the selectivity filter of the channel. On the other hand, the finding of big conductance jumps, suggesting simultaneous opening of multiple channels, is elusive in its underlying mechanism. Previously, using atomic force microscopy, we observed a clustering of channels in the membrane when the channels remain closed,29 and dynamic clustering and dispersion upon open-close transitions.30 These dynamic processes may underlie the simultaneous openings, in that nascent channels behave collectively even in the process of functional channel formation, although this issue needs to be studied further. There are several technical features in this study. First, we used microelectrodes for the electrophysiological recordings rather than AgCl wires immersed in droplets like in conventional DIB. The AgCl wires present a large surface area in the reconstituted IVTT solution, which is subject to electrochemical interference by various substances in the reconstituted IVTT, whereas the microelectrodes offer a stable junction potential, irrespective of the constituents. In addition, microelectrodes can be inserted into the droplets only when necessary for applying a membrane potential or current measurements. In the absence of a bilayer, such as in a separated droplet, microelectrodes are not inserted, and a pure isolated system is attained. Second, the use of a high concentration of azolectin dispersed in the hexadecane phase was another feature. In this study, adding lipids in the oil phase (lipid-out) rather than the aqueous phase (lipid-in)31 is a prerequisite to mimic the in vivo expression system since nascent channels should interact exclusively with the surface membrane of the DIB. If liposomes exist inside the IVTT droplet, the synthesized protein will interact with the liposomes before interacting with the surface membrane, and liposomeincorporated channels should transfer to the bilayer through liposome fusion to the DIB bilayer. This process is irrelevant to the physiological process of channel incorporation into the membrane. High concentrations of azolectin in the hexadecane phase offered a native environment for a first encounter of newly synthesized channels with the membrane, while substantially stabilized the bilayer. Apart from the technical features, our concurrent expression and functional detection system owes to a geometrical feature of DIB. The DIB provides a unique environment for synthesized proteins with a partial bilayer boundary. Once synthesized, the proteins reach the

interface of either the monolayer or bilayer, and eventually are inserted into the bilayer after freely diffusing on the surface across the monolayer−bilayer boundary. Even in the isolated droplet, the monolayer provides a pertinent environment for the close interaction of channel proteins, rescuing the synthesized proteins from aggregation (Figure 1A). Physically, the first appearance time represents the first passage time taken for the underlying multiple steps toward the appearance of a functional channel,32 which involves partitioning to the membrane interface, bilayer insertion, folding, and oligomerization. Even though the membrane potential is not a prerequisite for channel expression, as previous studies using liposomes in the absence of membrane potential showed massive expression, the finding of accelerated functional expression at higher membrane potentials of either polarity is surprising in two respects. First, the membrane potential dependency is physiologically relevant to in vivo bacterial syntheses, distinguishing the bacterial system from eukaryotic counterparts that operate at a nearly null potential. Second, the membrane potential dependency provides insights into underlying physical mechanisms of membrane insertion. It has been established that the hydrophobic membrane-spanning α-helix distributes at the membrane interface before being inserted.1 In the case of the KcsA channel, the biochemical experiments revealed that the channels remained monomeric at the membrane interface and likely formed α-helices. The accelerated first appearance time by the increased magnitude of the membrane electric field suggests that changes in membrane physical properties may contribute to the channelmembrane interaction; in a higher electric field, the bilayer thins and the bilayer tension decreases.26,27 The step of membrane insertion is most likely to be rate-limiting for channel formation, and a lower membrane tension in the high physiological bacterial membrane potential likely allows for ready insertion of the channel protein. This in vitro concurrent expression and functional detection system allows for further examinations of the molecular mechanisms responsible for the insertion of channel proteins into membranes, and the subsequent steps toward the generation of its active form, from a kinetic point of view, at the single-molecule level. Also, this system is promising for studying other types of membrane proteins, such as receptors and transporters.

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METHODS KcsA Channel. In vitro protein synthesis of the KcsA channel21 was performed using a reconstituted IVTT11,12 (PUREfrex 1.0, Gene Frontier, Kashiwa, Japan). WT and noninactivating open-gated mutant (H25R/E71A/E118A) DNAs were cloned into the pET-EmrE-myc vector33 using the In-Fusion cloning kit (Clontech) from the plasmids pQEKcsA34 and pQE-KcsA (H25R_E71A_E118A), respectively. The DNA fragments, which were amplified by PCR using T7F (5′-TAATACGACTCACTATAGGG) and T7R (5′GCTAGTTATTGCTCAGCGG) primers, were used as templates for IVTT. For comparison, the mutant channel was also synthesized in vivo using E. coli,34 and was reconstituted into the liposome and used for membrane fusion to the DIB. The DIB Method. DIBs17,18 were formed by dropping 1 μL droplets of reconstituted IVTT solution into a drop-in-well chamber filled with hexadecane solvent (Figure 1B). Azolectin (P3644, Sigma-Aldrich, St. Louis, MO) was dispersed in hexadecane at 100 mg/mL. Two droplets were automatically in F

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Notes

contact at the bottom of the drop-in-well chamber. The system was set on the stage of an inverted microscope (IX73, Olympus, Tokyo, Japan), and the temperature of the system was regulated using a glass plate heater (TP-110NLR, Tokai Hit, Fujinomiya, Japan). Rather than using gel-coated AgCl wires,18 we used microelectrodes filled with 3 M KCl for the electrical recordings, which were inserted into the droplets after the formation of the DIB (Figure 1B). The tip of the electrode was 1−2 μm in diameter and the electrode resistance was ≪1 MΩ. Microelectrodes provide an accurate evaluation of membrane voltage irrespective of the composition of the solution, whereas the previously used gel-coated AgCl wires have an unpredictable sequestering of solution components to the electrode surface that may interfere with membrane potential measurements. In the present method, the application of membrane potential and measurements of single-channel currents are arbitrarily performed on demand by inserting the microelectrodes into the droplets. The electrodes offsets can be performed by inserting two electrodes into a droplet. The channel current was recorded using an amplifier (EPC800USB, HEKA, Lambrecht/Pfalz, Germany) through a low-pass filter with a cutoff frequency of 1 kHz, sampled at 5 kHz using a digitizer (Digidata1550A, Molecular Devices, Sunnyvale, CA) and stored on a personal computer using the pCLAMP software (Molecular Devices).35,36 Channel Synthesis and Its Localization and Oligomeric State. When necessary, the synthesized proteins were labeled with [35S]-methionine and analyzed by SDS-PAGE and autoradiography. Protein bands were detected using a Typhoon FLA 7000 biomolecular imager (GE Healthcare UK Ltd., Buckinghamshire, England). For localization assays, protein synthesis with reconstituted IVTT was performed in the presence of 13.8 mg/mL of liposomes composed of 100% azolectin. Liposomes were prepared using an extruder (Avanti Polar Lipids, Alabama) with a 100 nm filter in accordance with the manufacturer’s instructions. The liposomes remain in solution after centrifugation at 20 000g for 30 min at 4 °C. To reveal the localization of the channel, the IVTT solution was centrifuged, and the supernatant was subjected to SDS-PAGE and autoradiography. The band intensity was compared to that before centrifugation. An oligomerization state investigation was performed based on the observation that tetrameric channels do not dissociate into monomers even in the presence of 1% SDS, unless boiled.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Atsuko Uyeda for technical assistance. This work was supported in part by KAKENHI Grant numbers 16H00767 (TM) and 16H00759, 17H04017 (SO), and ImPACT project (TM).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00454.

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REFERENCES

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Corresponding Authors

*Tel: +81 66789 4172. Fax: +81 66789 7428. E-mail: [email protected]. *Tel: +81 77661 8306. Fax: +81 77661 7101. E-mail: oiki-fki@ umin.ac.jp. ORCID

Tomoaki Matsuura: 0000-0003-1015-6781 Shigetoshi Oiki: 0000-0002-8438-6750 G

DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

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DOI: 10.1021/acssynbio.7b00454 ACS Synth. Biol. XXXX, XXX, XXX−XXX