Compartmentalized Assembly of Motor Protein Reconstituted on

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Compartmentalized Assembly of Motor Protein Reconstituted on Protocell Membrane toward Highly-Efficient Photophosphorylation Youqian Xu, Jinbo Fei, Guangle Li, Tingting Yuan, and Junbai Li ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04747 • Publication Date (Web): 21 Sep 2017 Downloaded from http://pubs.acs.org on September 23, 2017

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Compartmentalized Assembly of Motor Protein Reconstituted on Protocell Membrane toward Highly-Efficient Photophosphorylation Youqian Xu,† ‡ Jinbo Fei,† Guangle Li,† ‡ Tingting Yuan† ‡ and Junbai Li*† ‡

†

Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface

and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China

‡

University of Chinese Academic of Sciences, Beijing 100049, China

*

Address correspondence to [email protected]

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ABSTRACT

Molecule assembly and functionalization of protocells have achieved a great success. However, the yield efficiency of photophosphorylation in the present cell-like systems is limited. Herein, inspired by natural photobacteria, we construct a protocell membrane reconstituting motor protein for highly-efficient light-mediated adenosine triphosphate (ATP) synthesis through layer-by-layer technique. The assembled membrane, compartmentally integrating photoacid generator, proton conductor and ATP synthase, possesses excellent transparence, fast proton production and quick proton transportation. Remarkably, these favorable features permit the formation of a large proton gradient in a confined region to drive ATP synthase to produce ATP with high efficiency (873 ATP s-1). It is the highest among the existing artificial photophosphorylation systems. Such a biomimetic system provides a bioenergy-supplying scenario for the early photosynthetic life, and holds a promise in remotely-controlled ATP-consumed biosensor, biocatalysis and biodevices.

KEYWORDS: layer-by-layer assembly･biomimetic synthesis･motor protein･photoacid generator･ photophosphorylation


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Bioinspired molecule assembly has offered a great opportunity to construct dynamic functionalized nano- or microarchitectures, attracting much attention in nanoscience and nanotechnology.1-6 Assembled cell-like entities provide a fascinating window to understand the original form of life and develop advanced biodevices.7-9 Synthesis of protocells has achieved a great success. For instance, phospholipoprotein,10 protein-polymer,11 polymer and organic/inorganic hybrids12 have been assembled in a controlled manner to show various cell-like talents, such as cytoskeletal-like structures,13 predatory behavior14 and self-proliferating.15-17 However, bioenergy-production systems, as one of the key issues, have not yet received much attention. Photophosphorylation, forming ATP using the energy of sunlight, provides chemical energy within cells for most of metabolisms. Chemiosmotic theory has demonstrated that ATP synthesis by ATP synthase is coupled by a proton gradient across the phospholipid membrane.18-24 In modern life systems, through chemical oxidation or water splitting, respiration and photosynthesis create proton gradients to drive ATP synthase to produce ATP.25-28 However, it is still obscure to build a proton gradient in the prebiotic chemistry.29

Light is the primary energy source of life, which dramatically changes the chemical composition of the earth to promote life evolution.30 For mimicking natural photophosphorylation, a series of exciting artificial systems were constructed to capture light and move protons across the membranes.30-36 Bacteriorhodopsin (BR), is a membrane-embedded protein that can actively "pump" proton from one side of the membrane to the other, against an electrochemical gradient. After BR is incorporated into artificial phospholipid membrane, under irradiation, vectorial proton movement occurs to form proton gradient to propel ATP synthase to produce ATP.34, 35 Nevertheless, restricted by the efficiency of BR, the yield efficiency is limited. Therefore, it remains a great challenge to fulfill the requirement of ATP-driven bioapplications.37-39


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Scheme 1. Schematic illustration of the architecture of the multilayered protocell membrane. Under light illumination, as photochemical acid generator, PyOH uploaded by the PAH/GO film is dissociated into PyO- and H+. With the assistance of Nafion, light-mediated proton is conducted to form a proton gradient for driving ATP synthase to synthesize ATP in the presence of Pi and ADP. The ATP synthase structure is copied from RCSD PDB.

Herein, we present a bioenergy-supplying scenario by rationally engineering a multilayered protocell membrane to realize light-driven ATP synthesis with high efficacy (Scheme 1). The detailed assembly process is illustrated in Scheme 2. First, through simple and efficient layer-by-layer (LBL) assembly technique, driven by electrostatic interaction, poly (allylamine hydrochloride) (PAH) and graphene oxide (GO) are used as building blocks to construct the (PAH/GO)n film on a quartz slide via electrostatic interactions. Next, as one kind of polyaromatic derivatives possibly existing in the early earth, 1-hydroxy pyrene (PyOH) molecules are chosen as photoacid generators (PAGs) to be uploaded by the multilayered film via π−π stacking interaction with GO. Then, Nafion, as proton conductor, is coated on the surface of the abovementioned film. Finally, ATP synthase-proteoliposomes are spread to build up the artificial membrane with ACS Paragon Plus Environment

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well-organized structure. Under illumination, PyOH in the multilayered membrane is deprotonated quickly to release proton. Then, proton diffuses across the conductive film fast to form an efficient proton gradient and impulse ATP synthase to produce ATP with high efficiency.

Scheme 2. (A) Molecular formula of GO, PAH and PyOH; Schematic illustration of (B) the deposition process of (PAH/GO)n-PyOH on quartz slides and (C) the corresponding simplified molecular assembly (D) Schematic illustration of spin coating Nafion on (PAH/GO)n-PyOH; (E) Schematic

illustration

of

the

deposition

process

of

CFoF1-proteoliposomes

on

(PAH/GO)n-PyOH-Nafion and (F) the relevant simplified molecular assembly of the final membrane.


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RESULTS AND DISCUSSION

Purification of the Chloroplast FoF1-ATP Synthase. The chloroplast FoF1-ATP synthase (CFoF1-ATP synthase) was isolated and purified from fresh spinach, according to the previous reported methods with minor modifications.40 Figure 1A shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified CFoF1-ATP synthase, clearly revealing the existence of a, b and c subunits in the intramembrane Fo complexes, α, β, γ, δ and ε subunits in the soluble F1 part. Liposomes containing 10 wt% dimyristoylphosphatidylglycerol (DMPG) and 90 wt% dimyristoylphosphatidyl choline (DMPC) were added in a Triton X-100 solubilized CFoF1-ATP synthase buffer solution (10 mM Tricine-NaOH, pH 8.0, 20 mM NaCl and 5 mM MgCl2), allowing the protein-detergent micelles to incorporate. CFoF1-ATP synthase proteoliposomes were obtained after the removal of Triton X-100 with Bio-beads SM-2. The CFoF1 concentration is about 1.16 µM, which was measured by the Bradford method (Figure S1). Also, the bioactivity of reconstituted ATP synthase was determined by pH-jump induced ATP synthesis (Figure 1B).41 Based on the result above, the ATP synthesis rate in this case is about 180 s-1.

Figure 1. (A) SDS-PAGE analysis of the purified CFoF1-ATP synthase. (B) Changes of ATP concentration before and after the introduction of ATP synthase. After 50 µL of luciferin/luciferase was added into 150 µL of basic buffer (pH 8.8, 200 mM Tricine-NaOH, 2 µM ADP, 10 mM ACS Paragon Plus Environment

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NaH2PO4, 2.5mM MgCl2) to form Solution A, photon counter was recorded by using a BPCL ultra weak luminescence analyzer. 20 µL of DTT-reduced ATP synthase-liposome was mixed with 100 µL of acidic buffer (10 mM NaH2PO4; 2.5 mM MgCl2; pH 4.5) and co-incubated for 2 h to form Solution B. Then Solution A and Solution B were mixed together and photon counter was recorded to determine the activity of ATP synthase.

LbL Assembly of Protocell Membranes. LbL assembly has been a very versatile technique with good reproducibility for preparing the multifunctional films at nano- and microscale.42-44 In our case, negatively-charged GO (-41.9 mV) and positively-charged PAH (+32.0 mV) are chosen to construct the multilayered films on the quartz substrate in a controlled manner (Scheme 2B and 2C). Obviously, the characteristic absorbance of GO at 232 nm increases in a linear manner, indicating each assembled bilayer is deposited homogeneously (Figure 2A and 2B). For the (PAH/GO)6 film, the content of PyOH uploaded is up to 3.8×10-3 mmol cm-2 (Figure 2C and 2D). Then, clearly, the obtained slide becomes opaque, with the transparency of 14% (Figure 3A). Figure 3A show that sheet-like nanostructures appear on the substrate because of phase transformation-induced crystallization of PyOH, which has very limited dissolvability in water.45 It may halt following photochemical reaction due to the remarkable light scattering. Also, the rough surface may inhibit the spread of proteoliposomes membrane.46


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Figure 2. (A) UV-Vis spectra of multilayered (PAH/GO)n films; (B) the corresponding absorption of GO at 232 nm depended on the assembly layers; (C) UV-Vis spectra of (PAH/GO)n-PyOH films; (D) the relevant PyOH quantity in (PAH/GO)n-PyOH films.

Accompanying the introduction of Nafion, it is found that the transparency increases up to 90% (Figure 3B). The reason may be that Nafion-dissovled 1-propanol redissolve PyOH and effectively reduce the size of PyOH microcrystals. The excellent transparency of the system favors the following photochemical reactions. Meanwhile, the surface roughness thereof is obviously decreased. Smooth surface might contribute to the spread of proteoliposomes membrane. The entire membrane presents typical layer-like structure across section. In addition, comparing to the UV-Vis spectrum of PyOH ethanol solution, the much broader and red-shift absorption profile of (PAH/GO)6-PyOH appears (Figure S2). It may result from the molecular aggregation of PyOH.


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Figure 3. Optical images and SEM images of the surface and cross section of (A) Quartz-(PAH/GO)6-PyOH;

(B)

Quartz-(PAH/GO)6-PyOH-Nafion;

(C)

Quartz-(PAH/GO)6-PyOH-Nafion-CFoF1-Proteoliposomes. The use of the logo above is permitted by the Institute of Chemistry, Chinese Academy of Sciences (ICCAS). After the CFoF1 proteoliposomes are spread on the Nafion coated the multilayer film containing PAGs, the protrusions exist on the surface, signifying membrane incorporation of CFoF1-ATP synthase (Figure 3C). Thus, the final protocell membrane is obtained. To preliminarily verify the structural stability of ATP synthase after immobilization, Fourier transform infrared (FTIR) spectroscopy was performed. As shown in Figure S3, there is no obvious difference in the FTIR spectra of ATP synthase before and after immobilization, in good consistence with the previous report.47 The result reveals that immobilized biological motors remain stable, which is the premise of the following bioactivity of ATP synthesis. Furthermore, atomic force microscopy (AFM) was used to confirm the membrane surface after the modification of CFoF1-ATP synthase. The topographic images reveal that CFoF1-ATP synthase embeds into phospholipid membrane and ACS Paragon Plus Environment

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spread on the above-mentioned substrate homogeneously (Figure 4A and 4B). CFoF1-ATP synthase is composed of two linked multisubunit complex, a hydrophobic membrane-embedded Fo part and a hydrophilic F1 part. Possibly, the observed protrusions are F1 distributed on the membrane surface, in good accordance with the previous report.48 Also, the difference of chemical compositions between phospholipids and protein is discriminated by their interactions with the probe. As complementary to the topography images, the relevant phase images provide revealing protein domain formation and localization at lipid phase boundaries (Figure 4C and 4D). Clearly, they also certify ATP synthase is well distributed on the surface. After a statistics analysis based on the recent report,48 the average number of protrusions per cm2 is estimated to 3.2×1010 (Figure 4E-H). Considering that the molecular weight of the CFoF1-ATP synthase is 570 kD,49 the mass counted the average number of protrusions is determined approximately 30 ng cm-2, very close to the previous report.48

Figure 4. (A, B) AFM topography and 3D images of the surface of the final motor protein reconstituted protocell membrane; (C, D) relevant AFM phase and corresponding 3D images; (E-H) ACS Paragon Plus Environment

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AFM images of three regions (500×500 nm2) randomly taken from E for estimating the average number of proteins protruding more than 10 nm. The statistical results in F, G and H are 88, 74 and 78, respectively. The average density of ATP synthase is N=80 per 500×500 nm2.

Light-Responsive Proton Release. To roughly evaluate the light-induced deprotonation capacity of PyOH (Figure 5A, ∆pKa=4.6 with illumination50) in the membranes with different layers, time-dependent pH variations in deionized water (1 mL, pH=6.3) were detected under irradiation (Xenon lamp, 150 W, 60 cm). The schematic diagram of the experimental setup is shown in Figure 5B. As a whole, the ∆pH value is heavily relying on the quantity of encapsulated PyOH. That is, it increases along with the assembled layer number (Figure 5C). For (PAH/GO)6-PyOH, it is up to 2.5. Kinetically, the pH is rapidly declining from 6.3 to 3.8 within 5 min, which demonstrates that PAGs in the multilayered membrane can be effectively deprotonated under light illumination. In contrast, under the same illumination, the system without Nafion coating exhibits little pH variation. In detail, pH declines only 0.1 within the beginning 10 min (Figure 5D). This might ascribe to aggregation-restricted deprotonation. Light scattering reduces the availability for photochemical reaction. The results indicate that high transparency facilitates the photochemical reaction, permitting the rapid responsiveness. Thus, it favors the following ATP synthesis, which is driven by an artificially generated ∆pH. At present, we have to admit that it is a challenge for us to give the precise determination of light-induced ∆pH in such confined area. At the same time, it is difficult to use a fluorescent probe to test true pH changes between the inside and the outside, due to the distraction of hydrophobic PyOH with strong fluorescence in the assembled membrane.


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Figure 5. (A) Light-stimulated chemical reaction of PyOH; (B) Schematic illustration of the pH determination; (C) Time-dependent pH changes of (PAH/GO)n-PyOH-Nafion films (n=1, 3, 5, 6) under illumination; (D) pH changes of (PAH/GO)6-PyOH-Nafion and (PAH/GO)6-PyOH as a function of illumination time.

Light-Driven ATP Production. The activity of CFoF1-ATP synthase in the protocell membrane is evaluated by using the luciferin and luciferase assay to determine the ATP concentration as a function of time under dark or illumination. The schematic diagram of the experimental setup is shown in Figure 6A. It should be noted that during the illumination, the temperatures in our system had no obvious change. Under darkness, there is no ATP production yield in the whole process. As a comparison, under irradiation, the difference of outward proton concentration provides a proton gradient as a driving force for ATP synthase catalysis. In detail, the ATP production in 1 mL of complex buffer solution grows rapidly and reaches a plateau approximately 530 nmol L-1 after 120 s (Figure 6B). Noticeably, the plateau time in the system (120 s) is obviously shorter than ACS Paragon Plus Environment

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abovementioned light-induced deprotonation response in water (5 min, Figure 5D). It might be because the weak basic buffer solution (pH=8.0) could neutralize proton. In particular, in the beginning 5 s, the ATP production obtains a great speed to achieve 418 nmol L-1. During this time, the average CFoF1-ATP synthase turnover frequency (TOF) is about 873 ATP s-1. The detailed calculation is present in Supporting Information. To our best knowledge, it is the highest value among the existing light-responsive ATP synthesis systems (see Table 1).

Figure 6. (A) Schematic illustration of the home-made ATP production setup. The sample was immersed completely inside a 4 mm × 12 mm × 30 mm cell containing 1 mL of the buffer solution (pH=8.0, 10 mM Tricine-NaOH, 0.2 mM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2 and 30 mM NaCl). Note that, there was no temperature change in the quartz cell during the whole experiment. (B) Light-driven ATP synthesis on the multilayered protocell membrane without or with CCCP as an uncoupler.


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Table 1. Comparison of TOF in the existing light-driven ATP synthesis systems. Proton source

FoF1-ATPase source

Light source

TOF (s-1)

Ref.

Carotene-porphyrinnaphtoquinone

Spinach chloroplast

0.1 mW cm−2 (λ=633 nm)

7

31

BR

Bacillus PS3 cell

800 W (10 cm, 500-650 nm)

7

33

BR

Bacillus PS3 cell

5.04 W cm−2 (λ=570 nm)

3.2×10-2

34

BR

Bacillus PS3 cell

5.04 W cm−2 (λ=570 nm)

0.3

35

PAG

Spinach chloroplast

873

This work

150 W (60 cm, 200-1000 nm; 30 mW cm−2, λ=400 nm)

Indeed, ATP production is driven by proton gradient and membrane voltage difference. In our case, the driving force is not constant because of the dynamic photochemical reaction-mediated proton gradient. At present, we do not have the serviceable tool-set to monitor the real-time driving force in situ. Nevertheless, based on the result above, it could be assumed that the driving force in this case is much greater than those in these traditional systems. The possible reason can be ascribed to the special structure of the protocell membrane. On one hand, the mulitlayered membrane can effectively upload PAGs in a confined region that enhances localized concentration of protons to form a high proton gradient in a confined area. On the other hand, the rapid light response of PyOH greatly enhances the proton concentration in multilayered membrane in a short time. Thus, the ∆pH across the membrane might be much bigger, which brings a greater driving force for ATP production. Accompanying the photochemical reaction, proton source is gradually consumed and the driving force decrease, resulting in that ATP production is slowed down and even stopped (Figure 6B). As a comparison, ATP synthesis rates driven by acid-base transition (Table S1) generally showed higher values than those driven by light (Table 1).51-53 The reason can be explained that for previously reported light-driven systems, ATP synthesis rate was heavily restricted by the generation rate of “proton pump”. If the localized generation rate of the


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light-driven proton source is high enough, it can be envisioned that a larger proton gradient could be generated in such a system, leading to ATP synthesis with higher efficiency.

In contrast, a random system, (PAH/GO)mix-PyOH-Nafion-CFoF1-Proteoliposomes, was prepared as a control through simple co-precipitation of PAH and GO (Figure S4). As a result, under the same conditions, the corresponding TOF of ATP synthesis in the non-LbL mixture above is about 70 s-1 (Figure S5), which is much lower than that in an organized LbL membrane. In addition, another random system, (PAH/GO/PyOH/Nafion)mix-CFoF1-Proteoliposomes, was constructed with a considerably rough surface due to the existence of microfibers (Figure S6). Thus, it is hard for a transmembrane proton gradient to drive ATP synthase to synthesize ATP. The relevant TOF of ATP synthesis in this system is near to 30 s-1 (Figure S7). These findings suggest the importance of use of the LbL membrane for an organized system.

To prove that the increase of ATP concentration is definite due to the proton gradient-coupled ATP synthesis, we introduced an uncoupler (carbonyl cyanide-m-chlorophenylhydrazone, CCCP). CCCP is a proton ionophore that dissipates the proton gradient generated on the two sides of the membrane and thus inhibits phosphorylation.54-56 When CCCP was added to the system, no ATP was produced under illumination (Figure 6B). Note that, CCCP at this concentration has no obvious influence on the count of ATP yield, as shown in Figure S8. The result demonstrates that the process of ATP synthesis was primarily attributed to light-induced proton gradient-linked rotary catalysis of ATP synthase and only occurred in the presence of all components.

CONCLUSIONS

In summary, through compartmentally design and rationally construction, a bioinspired protocell membrane with multilayered structures is integrated via LBL assembly technique to achieve


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light-driven ATP synthesis with high efficiency. In the artificial membrane, via π−π stacking interaction with GO, PAGs are effectively uploaded with a high capacity of 3.8×10-3 mmol cm-2. The introduction of proton conductive Nafion into the system renders the film with excellent transparency (90%), which improves light utilization and promotes the photochemical reaction. Thus, under illumination, a large vectorial proton gradient forms quickly in a confined region to drive ATP synthase to produce ATP. The TOF is high up to 873 ATP s-1. It is the highest efficiency among the present light-driven systems of ATP synthesis. The assembled strategy will be helpful to understand energy supplying of the primary life. Such a biomimetic system holds a great potential in remotely-controlled ATP-consumed bioapplications.

METERIALS AND METHODS

Chemical Materials. Graphite, KMnO4 and NaNO3 were purchased from Sinopharm Chemical Reagent Co., Ltd. PyOH was bought from Aladdin. PAH were purchased from Sigma-Aldrich. Dimyristoylphosphatidyl choline (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were from Avanti in powder form. Nafion D-521 dispersion (5 wt% in water and 1-propanol, ≥0.92 meq/g exchange capacity) was bought from Alfa Aesar. β-D-octylglucoside, Tricine, MgCl2, NH4HCO3, dithiothreitol, Tris, Sodium dodecyl sulfonate (SDS), tetramethylethylenediamine (TEMES), acrylamide, were purchased from Sigma-Aldrich. Bovine serum albumin (BSA), Protein Marker (11-150 kDa) and Coomassie brilliant blue G250 were bought from Solarbio. Bio-beads SM-2 was purchased from Bio-Rad. Deionized water (specific resistance of 18.2 MΩ) was used in all experiments.

Preparation of GO. GO solution was obtained from graphite powder using a modified Hummers method.57 Typically, 1.0 g graphite powder was added to 70 mL H2SO4 (98%) and stirred at ambient temperature. 1.5 g NaNO3 was added and the mixture solution was kept in an ice bath. With ACS Paragon Plus Environment

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vigorously stirring, 9.0 g KMnO4 was added very slowly to keep the temperature below 10 °C. After stirred for 2 h, the mixture was put at room temperature and kept stirring overnight. Then, 140 mL of water was added to the mixture and stirred for 15 min. The mixture solution was further diluted by 500 mL water under vigorous stirring. 20 mL H2O2 (30%, wt) was then added to the mixture and the color of the solution changed from dark brown to brilliant yellow with bubbling. The mixture was centrifuged and washed with 250 mL of 1:10 HCl/water solution (V/V) and water by centrifugation until the pH of the suspensions was between 6 and 7. Finally, the product was dialyzed for 1 week to remove impurity and then freeze-dried for further use.

Isolation of Chloroplast FoF1-ATP Synthase. Chloroplast FoF1-ATP synthase was isolated and purified from spinach according to previous literature.40 In detail, a total of 200 g spinach leaves were washed, keeping at 4 °C overnight, and then homogenized in a blender, together with 500 mL of homogenization buffer B1 (0.4 M sucrose, 100 mM Tris-NaOH pH 8.0, 2 mM MgCl2). The leaf fragment was centrifuged at 10 600 rpm for 30 min to collect intact and broken chloroplasts. The precipitant was suspended in hypotonic buffer B2 (10 mM Tris-NaOH pH 8.0, 0.5 mM MgCl2) and stirred for 15 min to break the intact chloroplast. Following centrifugation at 16 900 g for 15 min, the pellet was suspended in high ionic strength buffer B3 (0.4 M sucrose, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM MgCl2) and stirring for 15min. The washed thylakoid membranes were centrifuged at 16 900g for 25 min. The supernatants were removed and resuspended by B4 (0.4 M sucrose, 50 mM Tricine-NaOH pH 8.0, 2 mM MgCl2). The chlorophyll concentration of thylakoid membranes suspension was determined and then adjusted to 5 mg Chl ml-1 using buffer B4. Solid dithiothreitol (50 mM final concentration) was added and the suspension was stirring 15 min. Then mixed an equal volume of extraction B5 (0.2 M sucrose, 20 mM MES-NaOH pH 6.5, 5 mM MgCl2, 400 mM (NH4)2SO4, 2 mM Na2-ATP, 50 mM DTT, 60 mM β-D-octylglucoside, 25 mM Na cholate) and gently stirred for 30 min at 4 oC. Then separated from the membranes by centrifugation at 208 ACS Paragon Plus Environment

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000 gmax for 60 min. The supernatant enriched CFoF1 protein was extracted by (NH4)2SO4 (30-40%) and stirred 15 min. The precipitate was collected by centrifugation at 12 000 g for 10 min and resuspended at a high concentration with 5 mL buffer B6 (30 mM NaH2PO4-NaOH pH 7.2, 200 mM sucrose, 2 mM MgCl2, 0.5 mM Na2 EDTA, 4 mM dodecyl maltoside). The resulting solution was used immediately or shock frozen in liquid nitrogen in Eppendorf tubes. The next sucrose density gradients (20, 28, 36, 44, 52 and 60% w/v) containing CFoF1 protein centrifuged at 242 000 gmax for 14 h at 4 oC. The CFoF1 protein complex migrates into the 44% sucrose layer and shock frozen in liquid nitrogen. The fractions of CFoF1-ATP synthase were analyzed by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gels. 10 µL of protein solutions were added to each line of the gel. After reconstitution into the proteoliposome, the activity was evaluated by pH-jump induced ATP synthesis.41

Reconstitution of CFoF1-ATP Synthase Liposome. Liposomes were prepared from a mixture of DMPC and DMPG (9:1 m/m). The reconstitution of CFoF1-ATP synthase into liposomes was described previously. Briefly, liposomes have been added in a Triton X-100 solubilized CFoF1-ATP synthase buffer solution (10 mM Tricine, pH 8.0, 20 mM NaCl and 5 mM MgCl2), allowing the protein-detergent micelles to incorporate, followed by three slow removal cycles of Triton X-100 by Bio-beads SM-2. This leads to the formation of CFoF1-ATP synthase proteoliposomes. The final concentration of lipid was 5 mg/mL. The protein concentration was determined by the Bradford method according to the previous report.58 For the calibration curve, 60 µL of 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL BSA standard solution in centrifuge tubes with the volume of 5 mL was mixed with 3 mL of coomassie brilliant blue G250, respectively. After 20 min, the absorbance of each sample at 595 nm was recorded (Figure S2). In this case, the relevant absorbance of CFoF1-ATP synthase-liposomes at 595 nm is 1.61. The final concentrations of CFoF1-ATP synthase and lipid were corrected as 1.16 µM and 5 mg/mL, respectively, although the actual concentration of ACS Paragon Plus Environment

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CFoF1-ATP synthase will be lower due to due to the low purity.

LBL Assembly of the Multilayered Films. The quartz substrates were cut into 15×6 mm rectangular pieces, cleaned in chromic acid lotion and H2O2/H2SO4 solution, finally rinsed with water. Positively charge PAH was first absorbed on the substrates by immersing PAH (10.0 mg mL-1) for 10 min. Then, after washing three times with water, the substrates were immersed into negative charge GO solution and washing three times with water. Thus, a pair of layers was obtained and this process was repeated until the expected pairs achieved. The obtained substrates were immersed into PyOH ethanol solution (10 mg mL-1) over 10 min, then put into water to quickly solidification of PyOH and dried at 50 °C. 50 µL of Nafion solutions (5 wt%) were spin onto

substrates

and

dried

at

50

°C.

The

deposition

of

CFoF1-liposome

on

the

(PAH/GO)n-PyOH-Nafion layer was carried out according to a method previously reported.49 In detail, in a buffer solution (pH 8.0, 20 mM Tricine-NaOH, 40 mM NaCl, 5 mM MgCl2), CFoF1-ATP synthase was immobilized by dipping a Quartz-(PAH/GO)n-PyOH-Nafion multilayered film in the CFoF1-ATP synthase-liposome suspension at room temperature in dark. After 2 h, the saturated amount of CFoF1-ATP synthase-liposome deposited on the coated quartz slide was achieved. Next, the assembled membrane was carefully washed with the buffer solution above for three times. The random system, (PAH/GO)mix-PyOH-Nafion-CFoF1-Proteoliposomes was also prepared as a control through simple co-precipitation of PAH and GO. Another random system, (PAH/GO/PyOH/Nafion)mix-CFoF1-Proteoliposomes, was also constructed as a control through simply mixing PAH, GO, PyOH and Nafion.

pH Determination. To measure the system pH value at different time with different layers to get the ∆pH value, the sample was added into the cell with 1 mL of water (initial pH0=6.3). Under illumination, time-dependent pH value (pHt) in water was recorded by using a commercial pH ACS Paragon Plus Environment

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meter (Mettler-Toledo FE-20K, 3 mm Ultra-Micro electrode).

Evaluation of ATP Synthesis. ATP production was measured by a bioluminescence assay (ENLITEN ATP Assay System, Promega, USA) using a BPCL ultra weak luminescence analyzer (BPCL-GP15, Institute of Biophysics, Chinese Academy of Sciences). Ahead 2 h prior to measurement, dithiothreitol was added to the proteoliposomes to a final concentration of 50 mM in order to allow the reduced enzyme. The motor protein reconstituted membrane mentioned above was immersed into 1 mL of the complex buffer solution (pH 8.0, 10 mM Tricine-NaOH, 0.2 mM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2 and 30 mM NaCl). The light source is a Xe lamp (HAMAMAZU, model E7536, P150 W). Before exposure and at subsequent time points, 5 µL of aliquot was taken from the reaction system and added with 50 µL of luciferin and luciferase to quantify the amount of ATP production as the function of time.

Characterization. Zeta-potential was measured by Zetasizer Nano ZS ZEN 3600 (Malvern). The UV-Vis absorption spectra were recorded with a UV-2600 (Shimadzu, Japan) spectrometer in water with 1 cm quartz cell at ambient temperature. SEM images were taken with an S-4800 scanning electron microscope, and the samples were allowed to dry by vacuum drying, followed by sputtering a thin layer of gold. AFM images were collected by FASTSCANBIO (Bruker) in a tapping mode. ATP was measured by a bioluminescence assay (ENLITEN ATP Assay System, Promega, USA) using a BPCL ultra weak luminescence analyzer. FTIR spectra were recorded by using a TENSOR27 instrument (Burker, Germany) with the help of calcium fluoride plates.

SUPPORTING INFORMATION

(1) The standard calibration curve of BSA; (2) UV-Vis absorbance spectra of (PAH/GO)6-PyOH, (PAH/GO)6-PyOH-Nafion and PyOH ethanol solution; (3) FTIR spectra of CFoF1-ATP synthase ACS Paragon Plus Environment

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liposomes before and after incubation with substrates; (4) Optical images and SEM images of the surface; (5) Light-driven ATP synthesis on the Quartz-(PAH/GO)mix-PyOH-Nafion-CFoF1 -Proteoliposomes

membrane;

(6)

Light-driven

ATP

synthesis

on

the

Quartz-(PAH/GO/PyOH/Nafion)mix-CFoF1-Proteoliposomes membrane; (7) The luminescence signal of CCCP at different concentrations; (8) The calculation of ATP produced efficiency. This material is available free of charge via the Internet at http://pubs.acs.org.

NOTES

Conflict of Interest: The authors declare no competing financial interest.

AUTHOR CONTRIBUTION Y. Xu and J. Fei contributed equally to this work.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the financial support for this research by the National Nature Science Foundation of China (No. 21433010, 21320102004, 21573248), the National Basic Research Program of China (No. 2013CB932802). J. F. particularly thanks the Youth Innovation Promotion Association of CAS (No. 2016032) and Institute of Chemistry, CAS (No. Y6290512B1).

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Compartmentalized Assembly of Motor Protein Reconstituted on

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