Application of Peptide Gemini Surfactants as Novel Solubilization Surfactants for Photosystems I and II of Cyanobacteria

Article pubs.acs.org/Langmuir

Application of Peptide Gemini Surfactants as Novel Solubilization Surfactants for Photosystems I and II of Cyanobacteria Shuhei Koeda,† Katsunari Umezaki,† Tomoyasu Noji,† Atsushi Ikeda,‡ Keisuke Kawakami,§ Masaharu Kondo,† Yasushi Yamamoto,† Jian-Ren Shen,∥ Keijiro Taga,† Takehisa Dewa,† Shigeru Ito,⊥ Mamoru Nango,§ Toshiki Tanaka,† and Toshihisa Mizuno*,† †

Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan § Graduate School of Science, Osaka City University, 3-3-138 Sugimoto-cho, Sumiyoshi, Osaka 558-8585, Japan ∥ Graduate School of Natural Science and Technology, Faculty of Science, Okayama University, Okayama 700-8530, Japan ⊥ Center for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan ‡

ABSTRACT: We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PGsurfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 °C compared with that using β-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV2+) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the lightirradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/ MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PGsurfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

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INTRODUCTION Photosystems I and II (PSI and PSII) are representative photosynthetic membrane proteins present in organisms from cyanobacteria to higher plants. They play important roles in absorbing photons from sunlight and producing electrons and holes via charge separation at the reaction center; the quantum yield is almost 1.0.1 Recently, the construction of artificial photosynthetic systems to produce hydrogen using solar energy has been examined by combining PSI with other reductases or reducing catalysts such as hydrogenases or platinum nanoparticles.1,2 A system for evolving oxygen gas from water via water splitting using solar energy has also been conducted by immobilizing PSII on inorganic materials such as gold nanoparticles3 or mesoporous silica.4 Constructing more sophisticated artificial photosynthetic systems5 consisting of multiple photosynthetic proteins with reducing catalysts or enzymes and electron mediators requires the design of an interface enabling intermediate and efficient photoinduced electron transfer and techniques for organizing such different components in desirable arrangements. © XXXX American Chemical Society

Recently, various peptide-containing surfactants consisting primarily of long alkyl chain and hydrophilic,6 β-sheet-forming,7 or the mixed amphiphilic8 peptide have garnered great interest owing to the unique properties arising from the peptide moiety. Subtle choices from a diverse sequence library enable us to finetune their assembled morphologies,9 for example, micelles, fibers, sheets, and gels, which are applicable to various biomaterials.10 Some peptide-based surfactants have been examined for use as solubilization surfactants for membrane proteins.11 McGregor et al. have reported that a surfactant composed of a 24-mer amphiphilic peptide with two long alkyl chains exhibits increased thermal stability via hydrophobic interaction between the transmembrane domain of membrane protein and the hydrophobic surface of amphiphilic peptide.12 Zhang et al. have reported a similar increase in thermal stability using an amphiphilic peptide surfactant, A6D.13 However, the Received: November 2, 2012 Revised: August 11, 2013

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Scheme 1. Synthesis of Asymmetric Viologen Containing Carboxyl Group

Synthesis of PG-Surfactant. Synthesis of each PG-surfactant was performed on a Rink-amide AM resin using commercially available Fmoc-protected amino acids, Fmoc-Cys(C12)-OH,14,17 and the standard condensation reagents (HOBT/HBTU/DIEA). The Nterminus of the PG-surfactant was end-capped with Ac2O. K3C12: HRMS (EI-TOF, [M + H]+): calcd. for C54H106N14O8S2, 1100.7667; found, 1100.7656. K4C12: HRMS (EI-TOF, [M + Na]+): calcd. for C60H117N13O9S2 + Na, 1250.8436; found, 1250.8412. DKDKC12: HRMS (EI-TOF, [M + H]+): calcd. for C56H104N11O13S2, 1202.7256; found, 1202.7253. DKDKC12K: HRMS (EI-TOF, [M + H]+): calcd. for C62H116N13O14S2, 1330.8206; found, 1330.8197. DKDKC12D: HRMS (EI-TOF, [M + Na]+): calcd. for C60H108N12O16S2 + Na, 1339.7345; found, 1339.7338. Synthesis of Monomethyl Viologen (1).18 To a solution of 4,4′bipyridyl (10 g, 64 mmol) in 100 mL of benzene, iodomethane (2.6 mL, 42 mmol) was added dropwise for 15 min (Scheme 1). The reaction mixture was stirred at room temperature for 48 h under a N2 atmosphere. After evaporation of the solvent, the obtained product was washed with excess DCM (100 mL × 5). From 1H NMR analysis, the purity of the target product was confirmed to be sufficient for the next step. Yellow solid (7.32 mg, 59%). 1H NMR (400 MHz, DMSOd6, rt) 4.39 (s, 3H, CH3−), 8.05 (d, 2H, pyridine-H-2′), 8.63 (d, 2H, pyridine-H-3′), 8.87 (d, 2H, pyridine-H-2), 9.15 (d, 2H, pyridine-H3). Synthesis of Asymmetric Methyl Viologen Containing a Carboxyl Group on One Side (2).18 To a solution of monomethyl viologen (2 g, 6.7 mmol) in 100 mL of MeOH, 3-bromopropionic acid (1.22 g, 8.0 mmol) was added (Scheme 1). The reaction mixture was refluxed for 36 h under a N2 atmosphere. The obtained precipitate was collected using a Hirsch funnel and washed with an excess of MeOH (100 mL × 3). 1H NMR analysis confirmed that the purity of the target product was adequate. Yellow solid (665.2 mg, 22% yield). 1H NMR (400 MHz, DMSO-d6, rt) 3.18 (t, 2H, CH2−COOH), 4.45 (s, 3H, CH3−), 4.91 (t, 2H, pyridine-CH2−), 8.79 (d, 2H, pyridine-H-2′), 8.81 (d, 2H, pyridine-H-2), 9.31 (d, 2H, pyridine-H-3′), 9.42 (d, 2H, pyridine-H-3). Synthesis of MV-DKDKC12K. The synthesis was performed on a Rink-amide AM resin using commercially available Fmoc-protected amino acids, Fmoc-Cys(C12)−OH, and the standard condensation reagents (HOBT/HBTU/DIEA). Instead of end-capping with Ac2O, 2 was condensed with a condensation reagent [HOBT/HBTU/DIEA in NMP/DCM (1/1)]. MV-DKDKC12K: HRMS (EI-TOF, [M + H]+): calcd. for C74H128N15O14S2, 1514.9215; found, 1514.9294. Determination of Critical Aggregation Concentration (CAC) of PG-Surfactants from Concentration-Dependent Profiles of Surface Tension. The surface tension of each PG-surfactant in a buffer at 30 ± 1 °C was measured using a static Wilhelmy plate with a dynamic contact angle tensiometer (DCAT 21, Dataphysics, Germany). To ensure the removal of surface-active contaminants, all glassware in contact with the sample was cleaned in chromic acid and rinsed with double-distilled water. The platinum Wilhelmy plate was washed in double-distilled water, heated on a Bunsen flame, and left to cool at room temperature. For the determination of the CAC value of

number of peptide-containing surfactants for the solubilization of membrane proteins remains limited. In a previous study,14 we reported novel peptide gemini surfactants (PG-surfactants) consisting of a hydrophilic oligoAsp peptide core ([Asp]n [n = 1−5]) and two peripheral dodecylamidomethyl-conjugated Cys residues (DnC12; n = 1− 5). Because the hydrophilic core peptide acts not only as a connector of the two dodecylamidomethyl chains but also as a polar headgroup, these surfactants are expected to have unique amphiphilic properties originating from the core peptide sequences. DnC12, which has an oligo-Asp core peptide, tends to form bilayer morphologies, especially for D3C12 and D4C12. If we choose other peptide sequences for these PG-surfactants, we obtain derivatives that function in other ways, for example, as solubilization surfactants for membrane proteins. In this study, therefore, we attempted to design PGsurfactants for use as solubilization surfactants for membrane proteins, especially PSI of Thermosynecoccus elongatus15 and PSII of Thermosynecoccus vulcanus.16 We sought to improve solubility and optimize through the screening of peptide sequences. We used spectroscopic observations and dynamic light scattering (DLS) and light-induced electron transfer activity measurements of PSI and PSII to study the effects of the designed PG-surfactants on their structures and functions. Further, we prepared a PG-surfactant derivative bearing the electron acceptor methyl viologen (MV2+) and evaluated it as a surface modifier to facilitate light-induced electron transfer from PSI to the MV2+ group conjugated to a solubilization surfactant moiety. Because the number of such multifunctional solubilization surfactants for membrane proteins remains limited, this study has the potential to offer a new strategy for treating membrane proteins as functional molecules in the design of artificial molecular devices.

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EXPERIMENTAL SECTION

Materials. All N-(9-fluorenyloxycarbonyl) (Fmoc)-protected Lamino acids, 1-hydroxybenzotriazole (HOBT), 2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), Rinkamide AM resin (200−400 mesh), N,N-diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), and N-methylpyrrolidone (NMP) were purchased from Merck Biosciences, Novabiochem (Switzerland), and Watanabe Chemical Industries (Japan). Dichloromethane (DCM) and methanol (MeOH) were purchased from Kanto Chemical Co., Inc. (Japan). 4,4′-Bipyridyl, iodomethane, 3-bromopropionic acid, 2-(N-morpholino)ethanesulfonic acid (MES), and tris(hydroxymethyl)aminomethane (Tris) were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Unless otherwise stated, other chemicals and reagents were obtained commercially and used without further purification. B

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each PG-surfactant, the concentration dependences of surface tension were measured. From the plot of log[C] vs surface tension and subsequent linear-fitting analyses, each CAC value was estimated. DLS and ζ-Potential Measurements of PG-Surfactant Assemblies. For the preparation of DLS samples of PG-surfactants, 5 μmol of a PG-surfactant was dissolved in 1 mL of 300 mM carbonate buffer (pH 10.0). The mean hydrodynamic diameters of the PGsurfactants in a 300 mM carbonate buffer solution (pH 10) at 25 °C were estimated using a Zetasizer Nano ZS (Malvern Instruments Ltd.) Purification of PSI of T. elongatus. Based on the method described in ref 19, a slightly modified method from that described in ref 5 was used. To solubilize PSI, thylakoid membranes of T. elongatus 20 (chlorophyll concentration 1.0 mg-Chl/mL) were incubated with a buffer [20 mM HEPES−NaOH (pH 7.2), 10 mM MgCl2, 25% (w/v) glycerol], containing 1 wt % β-n-dodecyl-Dmaltopyranoside (β-DDM), at 0 °C for 30 min in the dark. The extract was separated from the thylakoid membrane by ultracentrifugation (107 000g, 30 min). Anion-exchange chromatography (Toyo Pearl DEAE650S, TOSOH) performed with buffer C50 (30 mM MES− NaOH (pH 6.2), 0.03% β-DDM, 3 mM CaCl2, 0−500 mM NaCl) at 4 °C was used to isolate the PSI trimer. Increasing the salt concentration to 100 mM led to elution of PSI monomer and PSII. Increasing the salt concentration to 150 mM NaCl finally eluted trimeric PSI. The trimeric nature and purity of the PSI trimer were confirmed by BNPAGE as described in ref 21. The subunit composition was analyzed using SDS gel electrophoresis, as in ref 21. Purification of PSII of T. vulcanus. The PSII dimer complex was purified from cells of a thermophilic cyanobacterium, T. vulcanus, grown at 57 °C, as described previously.19,22 Thylakoid membranes isolated from cells were solubilized with 1 wt % (w/v) β-DDM. The solubilized mixture was passed through an anion-exchange chromatography column twice to purify the PSII dimer complex by separation from the PSI and PSII monomer complexes and other proteins. The obtained PSII dimer complex was suspended in 20 mM MES−NaOH (pH 6.0), 20 mM NaCl, 3 mM CaCl2, and 25% (w/v) glycerol, and then stored in liquid N2 until use. Replacement of Solubilization Surfactant from β-DDM to PG-Surfactants Using PEG Precipitation. Solutions of PSI trimer and PSII dimer after purification were diluted with 50% (w/v) PEG1450 (Sigma-Aldrich) to a final concentration of 17% (w/v) PEG1450 (Scheme 2). The PSI trimer and PSII dimer precipitates

(HA-50, Hoya, Saitama, Japan), and a 12-cm water layer illuminated a 1-cm diameter reaction vessel of volume 1 mL. A buffer containing 40 mM HEPES−NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, and 0.4 M sucrose, supplemented with 0.5 mM dichloroindophenol (DCIP) and 2 mM sodium ascorbate as an electron donor couple and 0.5 mM MV2+ as an electron acceptor was used for O2 uptake measurements. An aliquot of a sample solution was added to the buffer just before measurements. The concentration of PSI trimer solubilized with 0.006 wt % surfactant was 24 nM. The O2 uptake activity was estimated from the initial slope of the decrease in the O2 concentration upon illumination. O2 uptake from PSI solubilized with β-DDM and PG-surfactants under illumination lasted for about 5 min. Flash-Induced A698 Change of PSI after Heat Treatment. Flash-induced absorbance changes were measured using a split beam spectrophotometer at room temperature.23 The actinic flash was a 5 μs (half-width) pulse from a xenon flash lump of nearly saturating intensity (300 W). The reaction mixture contained 10 μM DCIP, 1 μM PMS, 10 mM sodium ascorbate, [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.8), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, ambient temperature. Signals (100 measurements) were averaged in each case. Evaluation of Photoinduced Electron-Transfer Rate of PSII from Increase in O2 Concentration. Measurements of O2 evolution activity of PSII were monitored at 25 °C using a Clark-type O2 electrode. The red-light source was the same as that described above. A buffer containing 40 mM MES−NaOH (pH 6.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, and 0.4 M sucrose was used for O2 evolution measurements. An aliquot of a sample solution was added to the buffer just before measurement. The concentration of PSII solubilized with 0.002 wt % surfactant was 32 nM. The exogenous electron acceptor used was 0.5 mM phenyl-p-benzoquinone. The O2 evolution activity was estimated from the initial slope of the increase in O2 concentration upon illumination. O2 evolution from PSII solubilized with β-DDM and PG-surfactants under illumination lasted for about 5 min. Fluorescence Spectrum at 77 K. The fluorescence spectrum of PSI was recorded using a fluorescence spectrophotometer (F-2500, Hitachi, Tokyo, Japan) with a laboratory-built liquid N2 Dewar flask at 77 K. PSI dispersed in a buffer, 40 mM HEPES−NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl2, and 15 mM MgCl2 at a concentration of 0.005 mg Chl/mL, which is a low enough concentration to avoid selfabsorption, was placed in a sample holder with a light path of 2 mm. The excitation wavelength for the fluorescence measurements was 430 nm. Preparation of Surface-Modified ITO Electrode and Immobilization of PSI Solubilized with MV-DKDKC12K. The ITO electrode was cleaned using a UV ozone cleaner (NL-UV253, Nippon Laser & Electronics Lab.) before surface modification. The cleaned ITO electrode was immersed in 100 μM HOOC-C5−SH (Dojin Chemical Lab.) in chloroform solution for 16 h, rinsed with chloroform, and then dried with N2 gas. The ITO electrode modified with COOH-terminated SAM was immersed in a PSI solution with MV-DKDKC12K (40 mM HEPES, 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, pH 7.5) for 16 h. After immersion, the ITO electrode was carefully rinsed with a buffer (40 mM HEPES, 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and pH 7.5), and then dried with N2 gas. The absorption spectrum of PSI immobilized on the ITO electrode was measured using a UV−vis spectrometer (UV-1800, Shimadzu) under dry conditions. Photocurrent Observations of PSI Immobilized on SurfaceModified ITO Electrode. Photocurrents were measured at −0.2 V (vs Ag/AgCl) in a laboratory-made cell that contained three electrodes: (1) PSI-immobilized electrode as the working electrode, (2) Ag/AgCl (saturated KCl) as the reference electrode, and (3) platinum flake as the counter-electrode. The solution consisted of 0.1 M phosphate buffer (pH 7.0) containing 0.1 M NaClO4. The incident monochromatic photon to photocurrent values ware determined using an SM-250 hyper-monolight system (Bunkoh-Keiki Co., Ltd., Japan).

Scheme 2. Surfactant Exchange from β-DDM to PGSurfactants

were collected by ultracentrifugation (104 000g, 30 min) at 4 °C. The precipitates of PSI trimer and PSII dimer were washed three times with fresh buffers (in the case of PSI trimer, 40 mM HEPES−NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2; in the case of PSII dimer, 40 mM MES−NaOH (pH 6.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2). The precipitated PSI timer and PSII dimer were resolubilized using 0.1 wt % (w/v) β-DDM or 0.1 wt % (w/v) PG-surfactant at 0 °C for 30 min in the dark. Evaluation of Photoinduced Electron-Transfer Rate of PSI from Decrease in O2 Concentration. Measurements of the O2 uptake activity of PSI were conducted at 25 °C using a Clark-type O2 electrode (Hansatech Instruments, DW1, Oxygen Electrode Unit, Norfolk, VA, United State). Red light from a 550-W halogen lamp through a red-pass filter (R-62, Hoya, Saitama, Japan), a heat-cut filter C

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RESULTS AND DISCUSSION Design of PG-Surfactants and Their Assembly Behaviors in a Buffer Solution. The basic chemical structure of PG-surfactants is illustrated in Figure 1. A hydrophilic short

measurement and other methods. Therefore, we designed the derivatives of DKDKC12 with an extra external amino acid on U-shaped molecular scaffold. Adding one Asp or Lys at the Nterminus of DKDKC12 yielded DKDKC12K and DKDKC12D, respectively (see Figure 1). These PG-surfactants displayed improved solubility at neutral pH and low CAC values. We attempted to determine CAC values using both the surface tension24 and the 8-anilino-1-naphthalene sulfonate (ANS) fluorescence methods.25 The observed changes in surface tension and ANS fluorescence at F472 in response to an increase in the concentration of DKDKC12K and DKDKC12D are illustrated in Figures 2 and 3, respectively. Zhang et al. have reported that unlike general single-chain surfactants such as sodium dodecyl sulfate, some gemini-type surfactants display a continuous decrease in surface tension even beyond their critical micelle concentrations after an increase in concentration.26 They inferred that such abnormal decrease in surface tension was related to an emergence of other complicated aggregates in addition to the formation of a surfactant monolayer at the air−water interface. Similar to the results in their system, DKDKC12K and DKDKC12D displayed continuous decreases in surface tension after the inflection point of surface tension (see Figure 2), and the CAC values based on the inflection point were estimated to be 5.0 μM for DKDKC12K and 0.37 μM for DKDKC12D. Conversely, the ANS fluorescence changes at 472 nm plateaued after their inflection points, and the CAC values based on these infection points were 8.3 μM for DKDKC12K and 7.9 μM for DKDKC12D, which differs greatly from determinations made using the surface tension method. To obtain other insights for the discussion of assembly behaviors of DKDKC12K and DKDKC12D in solution, we performed DLS measurements for DKDKC12K and DKDKC12D at various concentrations. The DLS profiles of DKDKC12K and DKDKC12D at 0.1 (ca. 1 mM) and 0.01 (ca. 0.1 mM) wt% are illustrated in Figures 4 and 5, respectively. At both concentrations, only a single fraction with a narrow distribution of hydrodynamic diameter at ∼6 nm was observed for both DKDKC12K and DKDKC12D, which indicated the formation of a micelle-type assembly. Because no aggregates larger than ∼6 nm were detected, even at concentrations above 0.1 wt % (ca. 1 mM), the formation of aggregates predicted from the continuous decrease in surface tension post-CAC seems to be a specific phenomenon at the air−water interface and is not the main species in the bulk solution. Therefore, the determination of CAC value from the ANS fluorescence assay seems suitable in our study. These data together with those of DnC12 (n = 3, 4) at pH 109 and KnC12 (n

Figure 1. Chemical structure of the peptide gemini surfactant (PGsurfactant). MV, methyl viologen.

peptide consisting of three or four amino acids is sandwiched by two dodecylamidomethyl-conjugated Cys residues via amide bonds. In a previous study, we found that three or four residues are likely suitable as a hydrophilic core peptide to lower the critical aggregation concentration (CAC).14 With the aim of improving solubilization at neutral pH, we first examined PGsurfactants with hydrophilic core peptides oligo-Lys (K3C12, K4C12), oligo-Arg (R3C12, R4C12), or oligo-Ser (S3C12, S4C12). Syntheses of all PG-surfactants was performed on a resin for Fmoc solid-phase peptide synthesis using the general Fmoc amino acids and the Fmoc-Cys-OH derivative, which has 1 dodecylamidomethyl group at the side chain via a thioether bond. Surprisingly, these surfactants were all insoluble at neutral pH. As in the Asp series of PG-surfactants (D3C12, D4C12), successive alignment of the same hydrophilic groups of the amino acids seems ineffective at providing adequate solubility at neutral pH. Therefore, we next prepared a PGsurfactant with an alternating sequence of acidic Asp and basic Lys as a core peptide (DKDKC12). Interestingly, unlike surfactants with successive alignments of Asp, Lys, Arg, and Ser, DKDKC12 became soluble at neutral pH but immediately turned insoluble, probably owing to charge neutralization within the molecule. The lower solubility of this precipitant prevented detail study of aggregation processes with DLS

Figure 2. Concentration dependence of the surface tensions of (a) DKDKC12K and (b) DKDKC12D in a 300 mM Tris HCl buffer (pH 7) at 30 °C. D

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Figure 3. Change in F472 of 8-anilino-1-naphthalene sulfonate (ANS) in accordance with increased concentration of DKDKC12K (a) or DKDKC12D (b). [PG-surfactant] = 0−1 mM, [ANS] = 10 μM, 20 mM phosphate buffer, 25 °C.

Table 1. Critical Aggregation Concentration (CAC) or Critical Micelle Concentration (CMC) Values for Surfactants at 30°C surfactant

CAC or CMC (μmol dm−3)

D3C12 D4C12 K3C12 K4C12 DKDKC12D DKDKC12K MV-DKDKC12K 12−6−12 β-DDM

3.0a 210a 5040b 11 500b 7.9c 8.3c 6.8c 1030d 170 000e

a

Data in ref 14. bObserved in 50 mM acetate buffer (pH 5). Observed in 50 mM phosphate buffer (pH 7). dData in ref 27. e Observed in water. MV, methyl viologen. β-DDM, β-n-dodecyl-Dmaltopyranoside. c

Figure 4. DLS profile of 0.1 wt % (red) and 0.01 wt % (blue) DKDKC12K in 20 mM phosphate (pH 7).

supramolecular pigment−protein complex; each PSI unit includes 12 protein subunits, 96 chlorophyll a (Chl a) molecules, and 3 [4Fe-4S] clusters. PSII of T. vulcanus16 is a dimeric supramolecular complex; each PSII unit includes 20 protein subunits, 35 Chl a molecules, and 1 Mn4CaO5 cluster. Crystallographic analysis of PSII has confirmed the binding of 12 lipid molecules per PSII unit.16 For treatment of these supramolecular membrane protein complexes with an aqueous buffer solution, the choice of solubilization surfactant is related to maintenance of the original quaternary supramolecular protein structures and functions. In this study, we designed novel solubilization surfactants DKDKC12K and DKDKC12D to solubilize PSI and PSII in a buffer and studied their effects on the original supramolecular structures and functions using a detergent-exchange method.28 First, we purified PSI and PSII following an established method19,22 using β-n-dodecyl-Dmaltopyranoside (β-DDM) as a solubilization surfactant. The purified PSI or PSII, solubilized with a buffer solution containing 0.1 wt % β-DDM was precipitated via the addition of poly(ethylene glycol) 1450,28 and the obtained precipitant was resolubilized with a buffer containing 0.1 wt % of either DKDKC12K or DKDKC12D. During resolubilization of the precipitants of PSI and PSII using both surfactants, we observed no residual aggregates. Because PSI and PSII bind various pigments such as Chl a and carotenoids in their protein frameworks, the comparison of absorption spectra is a good indicator of whether they can maintain the original supramolecular protein−pigment com-

Figure 5. DLS profile of 0.1 wt % (red) and 0.01 wt % (blue) DKDKC12D in 20 mM phosphate (pH 7).

= 3, 4) at pH 5 are summarized in Table 1. As a reference, the critical micelle concentration value of the cationic gemini surfactant 12−6−12,27 which consists of two quaternized ammonium head groups, two C12 alkyl chains, and a C6 alkyl chain linker, is also listed. Among these gemini-type surfactants with 2 C12 alkyl chains as a hydrophobic tail, DKDKC12K and DKDKC12D showed higher micelle formation properties. Solubilization of PSI and PSII Using PG-Surfactants DKDKC12K and DKDKC12D. PSI of T. elongatus15 is a trimeric E

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Figure 6. (a) Absorption spectra of photosystem I (PSI) solubilized with β-n-dodecyl-D-maltopyranoside (β-DDM; red) and DKDKC12K (blue); [PSI] = 24 nM, 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2, containing 0.1 wt % surfactant at ambient temperature. (b) Absorption spectra of PSI solubilized with β-DDM (red) and DKDKC12D (blue); [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2 containing 0.1 wt % surfactant at ambient temperature.

Figure 7. (a) Fluorescence spectra at 77 K of PSI solubilized with β-DDM (red line), DKDKC12K (green line), DKDKC12D (orange line), and 12− 6−12 (blue line); [PSI] = 24 nM, 40 mM HEPES (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1 wt % surfactant. (b) Chemical structure of cationic gemini surfactant 12−6−12.

Figure 8. (a) Absorption spectra of PSII solubilized with β-DDM (red) and DKDKC12K (blue); [PSII] = 32 nM, 40 mM MES (pH 6.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1 wt % surfactant at ambient temperature. (b) Absorption spectra of PSII solubilized with β-DDM (red) and DKDKC12D (blue); [PSII] = 32 nM, 40 mM MES (pH 6.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1 wt % surfactant at ambient temperature.

using β-DDM, indicating successful solubilization without loss of pigments such as Chl a and carotenoids (Figure 6b). To study more thoroughly the influence of DKDKC12K and DKDKC12D on the original membrane protein structure of PSI, we measured the fluorescence spectroscopy of PSI at 77 K.29 If free Chl a had been eliminated from the PSI protein framework, the fluorescence spectra at 680 nm would have emerged instead of that at 730 nm, originating from the red Chl a in the native PSI. The obtained fluorescence spectra of PSI solubilized with a buffer containing 0.1 wt % DKDKC12K and DKDKC12D at 77 K are illustrated in Figure 7a. The observed

plex. In this study, we used the absorption spectra of PSI and PSII solubilized in a buffer containing 0.1 wt % β-DDM as a control absorption spectrum of the native state because both protein complexes reported to maintain their original structures and functions when β-DDM is used. The absorption spectra of PSI solubilized with β-DDM and DKDKC12K are shown in Figure 6a. The obtained spectrum of PSI solubilized with DKDKC12K is completely consistent with that using β-DDM in the range 300−800 nm, indicating successful solubilization of PSI without denaturation. Similarly, the absorption spectrum of DKDKC12D showed perfect consistency with that obtained F

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Figure 9. DLS profiles of PSI solubilized with a buffer containing 0.1 wt % (a) or 0.001 wt % (b) β-DDM, DKDKC12K, or DKDKC12D; [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2.

fluorescence spectra using 0.1 wt % DKDKC 12K and DKDKC12D had a single peak at 730 nm and were consistent with that obtained using 0.1 wt % β-DDM, meaning that the protein−pigment complexes of PSI were maintained. By contrast, when we used 12−6−12 (Figure 7b) instead of DKDKC12K or DKDKC12D, the fluorescence spectra at 680 nm attributed to the elimination of Chl a was clearly observed. These data suggested that an appropriate linker must be chosen to apply gemini-type surfactants to membrane protein solubilization. The absorption spectra of PSII solubilized with 0.1 wt % DKDKC12K, and DKDKC12D are shown in Figure 8. Absorption spectra identical to those obtained using β-DDM as a solubilizer indicated successful solubilization of PSII with retention of its supramolecular pigment−protein complex. By contrast, if we used the reference cationic gemini surfactant 12−6−12 (see Figure 7b) as a solubilization surfactant, PSII failed to maintain its spectroscopic properties, as similar to PSI. Using DLS measurements, we examined whether our designed surfactants could solubilize PSI and PSII in singleprotein complexes. The DLS profiles of PSI solubilized with a buffer containing 0.1 or 0.001 wt % β-DDM, DKDKC12K, and DKDKC12D are shown in Figure 9. The DLS profiles of PSII solubilized with a buffer containing 0.001 wt % β-DDM, DKDKC12K, and DKDKC12D are shown in Figure 10. When we used 0.1 wt % surfactant, we detected only the DLS peak at less than 10 nm, which corresponds to the size of micelles of DKDKC12K and DKDKC12D owing to the large difference in concentration between PSI (19 nM) and each surfactant (0.1 wt %). However, using 0.001 wt % DKDKC12K or DKDKC12D, we observed the DLS peak only at the ca. ∼20 nm range, which corresponded to the size of PSI (21 nm, calculated from the Xray crystallographic analysis15), meaning that successful solubilization occurred without any aggregates, as shown in Figure 9b. By contrast, using 0.001 wt % β-DDM resulted in aggregates with hydrodynamic diameters greater than 1000 nm, probably owing to an unsatisfactory concentration; it is less than the CMC (0.15 mM [0.08 wt %] in water). Similarly, we observed only a single DLS peak at ca. 20 nm when using DKDKC12K and DKDKC12D for PSII solubilization, indicating that no PSII aggregates formed in solution.16 Therefore, compared with β-DDM, small amounts of our designed

Figure 10. DLS profiles of PSII solubilized with a buffer containing 0.001 wt % β-DDM, DKDKC12K, or DKDKC12D; [PSII] = 32 nM, 40 mM MES buffer (pH 6.5), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2.

DKDKC12K and DKDKC12D solubilized PSI and PSII, and even at such low concentrations both surfactants dispersed PSI and PSII in single-protein complexes in solution. To clarify whether our designed DKDKC 12 K and DKDKC12D surfactants could improve the thermal stability of PSI compared with that when using β-DDM, we next observed the time course of flash-induced absorption change at 698 nm. On applying short-time (∼5 min) heat treatment to PSI, Sonoike et al.30 have reported stepwise denaturation depending on temperature. When the supramolecular complex of PSI is maintained, the lifetime of the charge-separated state in which the electron moves to the FA/FB [4Fe-4S] cluster site in PsaC and the hole stays at the special pair (P700+•) in the PsaA/PsaB core dimer is observed ∼30 ms after flash light irradiation. When the PsaC subunit incorporating the FA/FB site is eliminated from the PsaA/PsaB core dimer owing to thermal denaturation, the other shorter lifetime at ∼1 ms emerges. Because electrons migrated to the FA/FB site are quenched by the dissolved oxygen with a certain possibility, concomitant longer lifetimes corresponding to the reducing process of P700+• via sodium ascorbate with the assistance of G

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Figure 11. Time-course flash-induced A698 changes of PSI solubilized with a buffer containing 0.1 wt % β-DDM (a), DKDKC12K (b), and DKDKC12D (c): [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1 wt % surfactant at ambient temperature. Flash excitation was applied using a xenon lamp (300 W, 5-μs pulse).

Table 2. Lifetime of P700+• of Photosystem I (PSI) Solubilized with a Buffer Containing 0.1 wt % β-n-Dodecyl-Dmaltopyranoside (β-DDM), DKDKC12K, or DKDKC12D at 25°C and after Heat Treatments at 60, 70, and 80°Ca surfactant β-DDM +•

lifetime of P700 (ms) (ratio of each lifetime fraction, %) before heat treatment after heat treatment at 60 °C

after heat treatment at 70 °C

after heat treatment at 80 °C

DKDKC12K denaturation degree (%)b

+•

lifetime of P700 (ms) (ratio of each lifetime fraction, %)

30 (60)

DKDKC12D denaturation degree (%)b

30 (37) 0

150 (40) 30 (63) 30

13 1 (13) 33 (30) 30 (37)

40 1 (40) 64 (21) 30 (33)

45 1 (45)

0 50 (51) 30 (57)

26

42

44 1 (44) 40 (19) 30 (40)

67 1 (67)

denaturation degree (%)b

30 (49)

1 (26) 48 (51) 30 (39)

1 (42) 100 (6) 30 (55)

lifetime of P700 (ms) (ratio of each lifetime fraction, %)

0 36 (63) 30 (23)

1 (30) 150 (7) 30 (52)

+•

60 1 (60)

Heat treatments were applied via incubation of PSI samples at 60, 70, and 80 °C for 5 min. After 15 min cooling at 4 °C, the time course flashinduced A698 changes, initiated by a xenon lamp flash (300 W, 5-μs pulse), were measured to estimate the decay rates of P700+•. [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, containing 0.1 wt % of surfactant. bDenaturation degrees were calculated by comparing the lifetime ratios at 1 ms (denatured state) and the sum of those at 30 ms and longer (native state). a

their ratios for each lifetime from a nonlinear curve fitting analyses of these time course curves of A698. The obtained data are summarized in Table 2. For all PSI samples solubilized with 0.1 wt % β-DDM, DKDKC12K, or DKDKC12D, the ratios of lifetime at ∼1 ms are negligible, meaning that no elimination of the FA/FB site occurred before heat treatment, an observation consistent with the results of our absorption and fluorescence spectroscopic analyses in this study. Heat treatments were applied by incubating each PSI sample solution at 60, 70, and 80 °C for 5 min. Before observation of the flash-induced A698 change at 25 °C, all samples were cooled at 4 °C for 15 min. The observed time course curves of A698 and the calculated lifetimes of P700+• and their ratios are summarized in Figure 11 and Table 2, respectively. Regardless of the surfactant or heat treatment applied, all ΔA698 values after light irradiation were

an electron mediator such as 5-methylphenazinium methylsulfate can be observed. Therefore, the population ratios of lifetimes at ∼1 ms (denaturation state) and the sum of other longer lifetimes (≥30 ms, native state) are a robust indicator of the degree of denaturation of the supramolecular quaternary structure of PSI. Before applying heat treatment, we first checked the time courses of flash-induced A698 changes of PSI solubilized with 0.1 wt % β-DDM, DKDKC12K, and DKDKC12D at 25 °C. The resulting decay curves of A698 are illustrated in Figure 11. On flash excitation with a xenon lamp (5 μs, 300 W), a transient decrease of A698 was observed, indicating P700+• generation. Recovery of A698 was observed after the charge recombination or direct reduction of P700+• by sodium ascorbate to recover steady-state P700. We calculated the lifetimes of P700+• and H

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similar, suggesting that P700 function is maintained through the current heat treatments. This result is consistent with that of previous studies.30 With 60 °C heat treatment, β-DDM (30% denaturation) and DKDKC12K (26% denaturation) showed similar degrees of denaturation, whereas DKDKC12D afforded slight resistance to thermal denaturation (13% denaturation). Although all surfactants showed a similar degree of denaturation with 70 °C heat treatment, at 80 °C, severe denaturation was observed using DKDKC12K and DKDKC12D (67 and 60% denaturation) compared with that using β-DDM (45% denaturation). Although slight improvement in thermal denaturation was observed for DKDKC12D on the 60 °C heat treatment compared with that accompanying β-DDM, the degree of improvement seems still low in these experiments. The results imply that the fundamental binding mode of β-DDM and the PG-surfactants DKDKC12K and DKDKC12D are similar where C12 alkyl chains contact the surface of the hydrophobic regions of PSI. Therefore, the peptide moieties of current PGsurfactants did not predominantly interact with the hydrophobic regions of PSI, which differs from the outcome using A6D or its analogs, which exhibits large thermal stability.31 Light-Induced Electron Transfer Activities of PSI and PSII Solubilized with β-DDM, DKDKC12K, or DKDKC12D. The effects of DKDKC12K or DKDKC12D solubilization on the light-induced electron transfer activities of PSI and PSII with were next examined. To evaluate the initial light-induced electron transfer rate in PSI, we chose an indirect assay system to monitor the consumption rate of dissolved oxygen in the presence of sacrificial reductant sodium ascorbate, the electron mediator dichloroindophenol (DCIP), and the electron acceptor MV2+.31 On light irradiation, the charge separation occurs first at the P700 special pair, and subsequently, the generated electrons migrate in accordance with the potential gradients through phylloquinone and the [4Fe-4S] cluster sites (Fx, FA, and FB) and are finally trapped in MV2+ molecules in a solution. Because the dissolved oxygen immediately quenches the one-electron reductant of MV2+, the initial rate of oxygen consumption can be considered identical to the initial rate of light-induced electron transfer in PSI. After the reduction of the resultant hole at the special pair (P700+•) by sodium ascorbate assisted by DCIP, one sequential electron transfer process initiated by light irradiation is completed. To calculate the initial rate of photoinduced electron transfer in PSI (24 nM), we added 2 mM sodium ascorbate, 0.5 mM DCIP, and 0.5 mM MV2+ to a PSI solution. The complicated minor electron transfer processes are concomitant in this assay system, requiring a careful experiment that considers background oxygen consumption. The oxygen decrease profile of PSI solution using DKDKC12K after light irradiation is illustrated in Figure 12. Although a background decrease in dissolved oxygen seems apparent even without PSI, a clear increase in oxygen consumption was observed in the presence of PSI. Electron transfer rates per single PSI unit were calculated from the background-subtracted oxygen consumption data, and the obtained initial rates are summarized in Table 3. The electron transfer rate per single PSI unit in a buffer containing 0.1 wt % β-DDM was calculated to be 39 PSI−1 s−1, which is comparable to that reported in previous studies.30 Conversely, using DKDKC12K and DKDKC12D instead of β-DDM resulted in no differences in the electron transfer process: the calculated rates were 42 PSI−1 s−1 for DKDKC12K and 41 PSI−1 s−1 for DKDKC12D. These data clearly indicated that PSI trimer could

Figure 12. Decrease in oxygen concentration in response to photoirradiation (from 620 to 720 nm) of PSI solubilized with DKDKC12K (orange line); [PSI] = 24 nM, 0.5 mM methyl viologen, 2 mM sodium ascorbate, 0.5 mM dichloroindophenol (DCIP), 40 mM HEPES (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, 0.4 M sucrose, and 0.1 wt % DKDKC12K. Green line is the reference data for the decrease in oxygen concentration in the absence of PSI.

Table 3. Light-Induced Electron-Transfer Rates in PSI and PSII Solubilized with β-n-Dodecyl-D-maltopyranoside (βDDM), DKDKC12K, or DKDKC12D solubilization surfactant β-DDM DKDKC12K DKDKC12D

surfactant concentration (wt %) 0.001 0.1 0.001 0.1 0.001 0.1

electron transfer rate in PSI (PSI−1 s−1)a

electron transfer rate in PSII (PSII−1 s−1)b

± ± ± ± ± ±

156 ± 0.9

41 39 42 42 44 41

2.1 1.2 0.7 1.0 1.5 2.3

153 ± 3.6 144 ± 4.9

a

Concentration of PSI solubilized with 0.1 or 0.001 wt % surfactant was 24 nM. A buffer containing 40 mM HEPES−NaOH (pH 7.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, and 0.4 M sucrose supplemented with 0.5 mM dichloroindophenol, 2 mM sodium ascorbate, and 0.5 mM MV2+ was used for oxygen uptake measurements. bConcentration of PSII solubilized with 0.001 wt % surfactant was 24 nM. A buffer containing 40 mM MES−NaOH (pH 6.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, 0.4 M sucrose, and 0.5 mM phenyl-p-benzoquinone was used for oxygen evolution measurements.

be solubilized by DKDKC12K or DKDKC12D without loss of light-induced electron transfer activity. To consider the influences of aggregation state of PSI on light-induced electron transfer, we observed the initial electron transfer rates using various concentrations of β-DDM, DKDKC12K, and DKDKC12D. Although this method was referred to the conditions reported in previous reports,31,32 the formation of PSI aggregates might occur under their conditions (0.0006 wt % β-DDM) and afraid to affect the accurate calculation of initial rates owing to the obstruction of the approach of substrates such as DCIP and MV2+. Our previous DLS observations revealed that PSI forms aggregates with 0.001 wt % β-DDM, and the average hydrodynamic diameter of these aggregates was higher than 1000 nm, as shown in Figure 8b. Such aggregates were also observed in the presence of 0.4 M sucrose. However, if we use 0.1 wt % β-DDM, PSI maintains monomeric dispersion. When DKDKC12K or DKDKC12D was used, PSI maintained a monomeric dispersion, even at a I

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evolution, we added 0.5 mM phenyl-p-benzoquinone as a sacrificial oxidant in the PSII (24 nM) solution. The calculated initial electron transfer rates per one PSII unit solubilized in a buffer containing 0.1 wt % β-DDM, DKDKC 12 K, or DKDKC12D are summarized in Table 3. Although slight decreases were apparent compared with results obtained using β-DDM, the initial electron transfer rates per PSII unit solubilized with 0.1 wt % DKDKC12K and DKDKC12D indicated maintenance. Similar to PSI, PSII can also be solubilized using DKDKC12K and DKDKC12D without loss of light-induced electron transfer activity. Derivatization of PG-Surfactant DKDKC12K for Novel Solubilization Surfactants to Trap Electrons from PSI. One of the benefits of including a peptide moiety within surfactant molecules is easy derivatization via the appending of various functional groups at the side or main chains of the peptide moiety. We next synthesized derivatives of DKDKC12K to trap the light-induced electrons to MV2+ in the outside of the PSI protein scaffold. MV2+ is among the best mediators for intervention in electron transfer35 from or to various proteins, and we appended it to the N-terminus of DKDKC12K (MVDKDKC12K) using MV2+ derivatives with ethyl carboxyl groups on one side.18 Concentrating MV2+ groups at the PSI periphery with the aid of amphiphilic interaction among the MV2+/ solubilization surfactant hybrid, DKDKC12K, and PSI might form a quasi-intramolecular interaction between MV2+ and PSI to promote effective light-induced electron transfer to the MV2+ group compared to outcomes when MV2+ is simply added to a solution (Figure 14). We calculated the CAC value of MV-DKDKC12K to be 6.8 μM using the ANS fluorescence method.25 This value is comparable to that of DKDKC12K (see Table 1). Solubilization of PSI using 0.1 wt % MV-DKDKC12K was performed in the manner used for DKDKC12K, and the absorption spectra are illustrated in Figure 15. Similar to the results with DKDKC12K, total consistency of the absorption spectrum to that with βDDM indicates that MV-DKDKC12K solubilizes PSI without

concentration of 0.001 wt % (see Figure 8b). We compared the initial electron transfer rate of PSI under these conditions (see Table 3). Interestingly, when 0.1 and 0.001 wt % of β-DDM, DKDKC12K, and DKDKC12D were used, the calculated initial electron transfer rates were similar, suggesting that this method is useful even in the presence of several PSI aggregates. Because this method was first applied to monitor the light-induced electron transfer activity of PSI embedded in thylakoid membranes33 and chloroplast34 dispersed in a buffer solution, these data may be reasonable. For PSII dimer, the electron transfer rate was calculated from the observed initial rate of oxygen evolution from the Mn4CaO5 cluster site (Figure 13).3 To saturate the initial rate of oxygen

Figure 13. Increase in oxygen concentration in response to photoirradiation (from 620 to 720 nm) of PSII solubilized with βDDM (red line) and DKDKC12K (blue line); [PSII] = 24 nM, 500 μM phenyl-p-benzoquinone, 40 mM MES (pH 6.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, 0.4 M sucrose, and 0.1 wt % surfactant.

Figure 14. Use of methyl viologen (MV2+)-appended DKDKC12K (MV-DKDKC12K) as a surface modifier for PSI to facilitate light-induced electron transfer. Sodium ascorbate works as a sacrificial reductant, and dichloroindophenol (DCIP) is an electron mediator between PSI and sodium ascorbate. J

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PSI to the MV2+ group in MV-DKDKC12K was observed in the presence of the sacrificial reductant sodium ascorbate (2 mM) and the electron mediator DCIP (0.5 mM). The electron transfer rates from PSI to MV2+ unit or MV2+ molecules were estimated from the initial rates of decrease in dissolved oxygen using the oxygen electrode, because the dissolved oxygen immediately quenches the one-electron reductant of MV2+. The amount of MV-DKDKC12K against PSI was set constant at 167 equiv to reduce the influence of free MV-DKDKC12K that did not bind to PSI. Because approximately 200 molecules of β-DDM were predicted to bind to one PSI, the equivalence of MV-DKDKC12K in this study seems reasonable. Increasing PSI concentration from 4 to 72 nM, increasing the concentration of MV-DKDKC12K proportionally from 2 to 12 μM. Although the concentrations of MV-DKDKC12K in this experiment were inadequate to maintain the monomeric dispersion of PSI (see Figure 16), the current indirect assay system is still useful for calculating electron transfer rates from the detailed consideration of aggregation influence of PSI described in the previous section (see Table 3). In the reference intermolecular system, the amount of MV2+ molecules against PSI was set at 167 equiv, as in the quasi-intramolecular system. The MV2+ molecules were added to the PSI solution solubilized with 0.1 wt % β-DDM. The initial rates of light-induced electron transfer are summarized in Figure 17. On decreasing

Figure 15. Absorption spectra of PSI solubilized with β-DDM (red) and MV-DKDKC12K (blue); [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.8), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2, containing 0.1 wt % surfactant at ambient temperature.

eliminating pigments. Using the DLS measurements, we studied PSI solubilized with 0.0001, 0.001, and 0.01 wt % MV-DKDKC12K, and the data obtained are illustrated in Figure 16. The use of 0.0001 wt % (6.7 μM) MV-DKDKC12K induced

Figure 16. DLS profiles of PSI (24 nM) solubilized with a buffer containing 0.01 wt % (red line, 670 μM), 0.001 wt % (green line, 67 μM), or 0.0001 wt % (blue line, 6.7 μM) MV-DKDKC12K; [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2 at ambient temperature. Figure 17. Concentration dependence of the MV2+ molecule or MVDKDKC12K on light-induced electron transfer from PSI to the MV2+ unit or molecules. The concentration ratio of MV2+ or MVDKDKC12K against a single PSI unit was set at 167. [PSI] = 12, 24, 36, 48, 72 nM, [MV2+ salt or MV-DKDKC12K] = 2, 4, 6, 8, 12 μM, [βDDM] = 0.1 wt %, 40 mM HEPES−NaOH (pH 7.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, 0.4 M sucrose, 0.5 mM DCIP, and 2 mM sodium ascorbate.

the formation of PSI aggregates with diameters greater than 1000 nm. Increasing the concentration to 0.001 wt % (67 μM) resulted in only a single DLS peak at ∼20 nm, suggesting monomeric dispersion of PSI. With respect to the solubilization efficiency for PSI, MV-DKDKC12K showed properties similar to those of DKDKC12K. We studied the concentration dependence of the MV2+ group or MV2+ molecules on the initial rate of light-induced electron transfer from PSI to the MV2+ moiety in MVDKDKC12K or MV2+ molecules in solution by examining the use of MV2+/surfactant hybrid MV-DKDKC12K as a solubilization surfactant with the supplemental addition of MV2+ molecules to a PSI solution. Because the MV2+ group in MVDKDKC12K should surround the periphery of PSI when MVDKDKC12K is used as a solubilization surfactant, we expected better efficiency in electron transfer between PSI and the MV2+ group in this quasi-intramolecular system compared with an intermolecular system in which MV2+ molecules are simply added in the PSI solution. Light-induced electron-transfer from

the concentration of MV2+ molecules, the electron transfer rate from PSI to MV2+ molecules showed monotonous decrease, suggesting that this process is a typical bimolecular reaction system. When the concentration of MV2+ decreased to 2 μM, the initial rate went down to ∼15 PS−1 s−1. Conversely, in the quasi-intramolecular system, MV-DKDKC12K sustained the initial rate at ∼23 PSI−1 s−1 irrespective of the decrease in MVDKDKC12K concentration, especially those less than 8 μM. Although the benefit of tethering the MV2+ group to DKDKC12K seems low owing to the lack of optimization of K

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Figure 18. (a) Adsorption of PSI solubilized with MV-DKDKC12K onto the modified gold electrode with 7-carboxy-1-hexanethiol. (b) Absorption spectra of PSI solubilized with MV-DKDKC12K in a buffer solution (blue line, [PSI] = 24 nM, 40 mM HEPES, pH 7.8, 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, 0.4 M sucrose, and 0.1 wt % MV-DKDKC12K) and onto a surface-modified indium tin oxide (ITO) electrode (red line). (c) Observation of photocurrent generation from PSI/MV-DKDKC12K immobilized on a carboxylate-modified ITO electrode with response to pulse irradiations of light at 440 nm. Before adsorption onto the ITO electrode, PSI was solubilized in a buffer containing MV-DKDKC12K. The ITO electrode was immersed in a buffer containing 40 mM HEPES (pH 7.8), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2. (d) Observation of photocurrent generation from PSI immobilized on a carboxylate-modified ITO electrode with response to pulse irradiations of light at 440 nm. Before adsorption onto the ITO electrode, PSI was solubilized in a buffer containing β-DDM. The ITO electrode was immersed in a buffer containing 40 mM HEPES (pH 7.8), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1 mM MV2+ salt.

After adsorbing PSI/β-DDM onto the carboxylate-modified ITO electrode, we observed the photocurrent by applying a negative potential (−0.2 V vs Ag/AgCl) to the ITO electrode in the presence of 0.1 mM MV2+ molecule as an electron mediator in an electrolytic solution. The direction of the lightinduced electric current (photocurrent) was cathodic in response to regular intervals of light irradiation at 440 nm, indicating that the P700 side was preferentially facing to the ITO electrode side, as shown in Figure 18d. On the other hand, because PSI/MV-DKDKC12K on the ITO electrode also yielded a cathodic photocurrent (Figure 18c), the P700 side of PSI is the preferential side to face the electrode side. The photocurrent showed faster responses with the use of MVDKDKC12K after light irradiation compared with that using βDDM+MV2+, and the quantum yield was 0.03%. These results imply that the solubilization of PSI with a surfactant/MV2+ hybrid such as MV-DKDKC12K plays a better role in lightinduced electron transfer from PSI both in solution and on an electrode.

the linker length between the DKDKC12K moiety and the MV2+ group within MV-DKDKC12K in this study, DKDKC12K and, likely, DKDKC12D are good molecular scaffolds that confer various desirable functions by simply tethering other functional groups at the side or main chain of surfactant molecules without the loss of the original functions of solubilizing membrane proteins. By adsorbing PSI solubilized with MV-DKDKC12K onto an indium tin oxide (ITO) electrode with a surface modified with 7-carboxy-1-hexanethiol, we examined photo current generation from PSI (Figure 18a).36 We attempted to immobilize PSI/MV-DKDKC12K using electrostatic interaction by mounting the PSI/MV-DKDKC12K solution (ca. 200 μL) onto the carboxylate-modified ITO electrode (1 cm × 1 cm) for 16 h at 4 °C. The nonspecifically binding species were washed away several times in a buffer solution. A comparison of the absorption spectrum of PSI onto ITO electrode with that in a buffer containing 0.1 wt % β-DDM showed that the immobilized PSI retained its original protein structure (Figure 18b). Previous studies of adsorption of photosynthetic membrane proteins onto electrodes have shown that this spontaneous adsorption method preferentially forms a protein monolayer37 rather than protein multilayer, which allows discussion of preferential membrane protein orientation onto the ITO electrode. If the P700 side of PSI is preferentially facing to the electrode side, the direction of light-induced electric current becomes cathodic because it is easier to supply electrons from the electrode to reduce the generated P700+•.

■

CONCLUSION We designed novel PG-surfactants, DKDKC 12 K and DKDKC12D, which can solubilize PSI and PSII in an aqueous buffer solution. Absorption and fluorescence spectroscopic properties revealed that DKDKC12K and DKDKC12D can solubilize PSI and PSII without eliminating pigments such as Chl a and carotenoids. With respect to the light-induced electron transfer activities of these membrane protein L

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(8) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-Assembly and Mineralization of Peptide-Amphiphile Nanofibers. Science 2001, 294, 1684. (9) de Loos, M.; Feringa, B. L.; van Esch, J. H. Design and Application of Self-Assembled Low Molecular Weight Hydrogels. Eur. J. Org. Chem. 2005, 17, 3615−3631. (10) Chen, J. X.; Wang, H. Y.; Li, C.; Han, K.; Zhang, X. Z.; Zhuo, R. X. Construction of surfactant-like tetra-tail amphiphilic peptide with RGD ligand for encapsulation of porphyrin for photodynamic therapy. Biomaterials 2011, 32, 1678−1684. (11) Zhao, X. Design of self-assembling surfactant-like peptides and their applications. Curr. Opin. Colloid Interface Sci. 2009, 14 (5), 340− 348. (12) McGregor, C. L.; Chen, L.; Pomroy, N. C.; Hwang, P.; Go, S.; Chakrabartty, A.; Prive, G. G. Lipopeptide detergents designed for the structural study of membrane proteins. Nat. Biotechnol. 2003, 21, 171− 176. (13) Koutsopoulos, S.; Kaiser, L.; Eriksson, H. M.; Zhang, S. Designer peptide surfactants stabilize diverse functional membrane proteins. Chem. Soc. Rev. 2012, 41, 1721−1728. (14) Umezaki, K.; Sakai, S.; Koeda, S.; Yamamoto, Y.; Kondo, M.; Ikeda, A.; Dewa, T.; Taga, K.; Tanaka, T.; Mizuno, T. Formation of Planar Bilayer Membranes on Solid Supports Using Peptide Gemini Surfactants. Chem. Lett. 2012, 41, 1430−1432. (15) Jordan, P.; Fromme, P.; Witt, H. T.; Klukas, O.; Saenger, W.; Krauû, N. Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution. Nature 2001, 411, 909−917. (16) Umena, Y.; Kawakami, K.; Shen, J. R.; Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 2011, 473, 55−60. (17) Lumbierrers, M.; Palomo, J. M.; Kragol, G.; Roehrs, S.; Müller, O.; Waldmann, H. Solid-Phase Synthesis of Lipidated Peptides. Chem.−Eur. J. 2005, 11, 7405−7415. (18) Oh, M.-K.; Bae, S.-E.; Yoon, J.-H.; Roberts, M. F.; Cha, E.; Lee, C.-W. Synthesis, Characterization, and Electrochemical Behavior of Viologen-Functionalized Poly(Amidoamine) Dendrimers. Bull. Korean Chem. Soc. 2004, 25, 715−720. (19) Shen, J.-R.; Kamiya, N. Crystallization and the Crystal Properties of the Oxygen-Evolving Photosystem II from Synechococcus Vulcanus. Biochemistry 2000, 39, 14739−14744. (20) Fromme, P.; Witt, H. T. Improved isolation and crystallization of Photosystem I for structural analysis. Biochim. Biophys. Acta 1998, 1365, 175−184. (21) Takasaka, K.; Iwai, M.; Umena, Y.; Kawakami, K.; Ohmori, Y.; Ikeuchi, M.; Takah, Y. Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium. Biochim. Biophys. Acta 2010, 1797, 278−284. (22) Kawakami, K.; Iwai, M.; Ikeuchi, M.; Kamiya, N.; Shen, J. R. Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and X-ray crystallography. FEBS Lett. 2007, 581, 4983− 4987. (23) Hoshina, S.; Sakurai, R.; Kunishima, N.; Wada, K.; Itoh, S. Selective destruction of iron-sulfur centers by heat/ethylene glycol treatment and isolation of Photosystem I core complex. Biochim. Biophys. Acta 1990, 1015, 61−68. (24) Osipow, L. I. Surface Chemistry: Theory and Industrial Applications; Reinhold: NewYork, 1962; p 188. (25) De Vendittis, E.; Palumbo, G.; Parlato, G.; Bocchini, V. Anal. Biochem. 1981, 115, 278−286. (26) Zhang, Q.; Gao, Z.; Xu, F.; Tai, S.; Liu, X.; Mo, S.; Niu, F. Surface Tension and Aggregation Properties of Novel Cationic Gemini Surfactants with Diethylammonium Headgroups and a Diamido Spacer. Langmuir 2012, 28, 11979−11987. (27) Danino, D.; Talmon, Y.; Zana, R. Alkanediyl-apbis(dimethylalky1ammonium bromide) Surfactants (Dimeric Surfactants). 5. Aggregation and Microstructure in Aqueous Solutions. Langmuir 1995, 11, 1448−1456. (28) Tavish, H. M.; Picorel, R.; Seibert, M. Stabilization of Isolated Photosystem II Reaction Center Complex in the Dark and in the Light

complexes, DKDKC12K and DKDKC12D can solubilize without the loss of original protein functions. Furthermore, DKDKC12D showed a slight improvement against thermal denaturation of PSI below 60 °C compared with that of β-DDM, as studied from the lifetime measurement of the light-induced chargeseparated state of PSI. By conjugating an electron acceptor, MV2+, we designed the surface-modified surfactant MVDKDKC12K to trap light-induced electrons from PSI both in solution and on an electrode. Although at present β-DDM is commonly used as an effective solubilization surfactant,38 especially for PSI and PSII, the discovery of other effective molecules that not only solubilize PSI and PSII without denaturation but also enable easy conjugation with various functional groups has the potential to open up new research avenues in this area. This demonstration of surface tuning of membrane protein functions by combining designed PGsurfactants with electron mediators demonstrates the versatility of our strategy. Application to membrane proteins, especially in relation to artificial photosynthesis, is ongoing in our group.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Young Scientists (B) No. 23750187 from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Adaptable and Seamless Technology Transfer Program through Targetdriven R&D, Japan Science and Technology Agency (JST).

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REFERENCES

(1) Lubner, C. E.; Grimme, R.; Bryant, D. A.; Golbeck, J. H. Wiring photosystem I for direct solar hydrogen production. Biochemistry 2010, 49, 404−414. (2) Ihara, M.; Nishihara, H.; Yoon, K.; Lenz, O.; Friedrich, B.; Nakamoto, H.; Kojima, K.; Honma, D.; Kamachi, T.; Okura, I. Lightdriven Hydrogen Production by a Hybrid Complex of a [NiFe]Hydrogenase and the Cyanobacterial Photosystem I. Photochem. Photobiol. 2006, 82, 676−682. (3) Noji, T.; Suzuki, H.; Gotoh, T.; Iwai, M.; Ikeuchi, M.; Tomo, T.; Noguchi, T. Photosystem II-Gold Nanoparticle Conjugate as a Nanodevice for the Development of Artificial Light-Driven WaterSplitting Systems. J. Phys. Chem. Lett. 2011, 2, 2448−2452. (4) Noji, T.; Kamidaki, C.; Kawakami, K.; Shen, J. R.; Kajino, T.; Fukushima, Y.; Sekitoh, T.; Itoh, S. Photosynthetic Oxygen Evolution in Mesoporous Silica Material: Adsorption of Photosystem II Reaction Center Complex into 23 nm Nanopores in SBA. Langmuir 2011, 27, 705−713. (5) Badura, A.; Esper, B.; Ataka, K.; Grunwald, C.; Wöll, C.; Kuhlmann, J.; Heberle, J.; Rögner, M. Light-Driven Water Splitting for (Bio-)Hydrogen Production: Photosystem 2 as the Central Part of a Bioelectrochemical Device. Photochem. Photobiol. 2006, 82, 1385− 1390. (6) Kiagus-Armad, R.; Brizard, A.; Tang, C.; Blatchly, R.; Desbat, B.; Oda, R. Cooperative and reciprocal chiral structure formation of an alanine-based peptide confined at the surface of cationic surfactant membranes. Chem.Eur. J. 2011, 17, 9999−10009. (7) Cavalli, S.; Handgraaf, J. -W.; Tellers, E. E.; Popescu, D. C.; Overhand, M.; Kjaer, K.; Vaiser, V.; Sommerdijk, N. A. J. M.; Papaport, H.; Kros, A. Two- Dimensional Ordered β−Sheet lipopeptide Monolayers. J. Am. Chem. Soc. 2008, 128, 13959−13966. M

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Article

Using Polyethylene Glycol and an Oxygen-Scrubbing System. Plant Physiol. 1989, 89, 452−456. (29) Mullet, J. E.; Burke, J. J.; Arntzen, C. J. Chlorophyll Proteins of Photosystem I. Plant Physiol. 1980, 65, 814−822. (30) Sonoike, K.; Hatanaka, H.; Katoh, S.; Itoh, S. Heat-stability of iron-sulfur centers and P-700 in Photosystem I reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus. Plant Cell Physiol. 1990, 31, 865−870. (31) Matsumoto, K.; Vaughn, M.; Bruce, B. D.; Koutsopoulos, S.; Zhang, S. Designer Peptide Surfactants Stabilize Functional Photosystem-I Membrane Complex in Aqueous Solution for Extended Time. J. Phys. Chem. B 2009, 113, 75−83. (32) Tiwari, A.; Jajoo, A.; Bharti, S. Heat-induced changes in photosystem I activity as measured with different electron donors in isolated spinach thylakoid membranes. Photochem. Photobiol. Sci. 2008, 7, 485−491. (33) Katoh, S. Studies on electron transport associated with photosystem I. II. Role of plastocyanin in methyl viologen photoreduction in French press-treated chloroplasts. Biochim. Biophys. Acta 1972, 283, 293−301. (34) Izawa, S. Acceptors and donors and chloroplast electron transport. Methods Enzymol. 1980, 69, 413−434. (35) Gomez, M.; Li, J.; Kaifer, A. E. Surfactant Monolayers on Electrode Surfaces: Self-Assembly of a Series of Amphiphilic Viologens on Gold and tin Oxide. Langmuir 1991, 7, 1797−1806. (36) Manocchi, A. K.; Baker, D. R.; Pendley, S. S.; Nguyen, K.; Hurley, M. M.; Bruce, B. D.; Sumner, J. J.; Lundgren, C. A. Photocurrent Generation from Surface Assembled Photosystem I on Alkanethiol Modified Electrodes. Langmuir 2013, 29, 2412−2419. (37) Kondo, M.; Nakamura, Y.; Fujii, K.; Nagata, M.; Suemori, Y.; Dewa, T.; Iida, K.; Gardiner, A. T.; Cogdell, R. J.; Nango, M. SelfAssembled Monolayer of Light-Harvesting Core Complexes from Photosynthetic Bacteria on a Gold Electrode Modified with Alkanethiols. Biomacromolecules 2007, 8, 2457−2463. (38) VanAken, T.; Foxall-Vanaken, S.; Castleman, S.; FergusonMiller, S. Alkyl glycoside detergents: synthesis and applications to the study of membrane proteins. Methods Enzymol. 1986, 125, 27−35.

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dx.doi.org/10.1021/la402167v | Langmuir XXXX, XXX, XXX−XXX