Effect of Lipid Phase Transition on Molecular Assembly and Structural

Nov 8, 2010 - Kamo, N.; Mitaku, S. Protein Eng. 1999, 12, 755-759; Yokoyama, Y.; Sonoyama, M.; Mitaku, S. J. Biochem. 2002, 131, 785-790). In order to...
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J. Phys. Chem. B 2010, 114, 15706–15711

Effect of Lipid Phase Transition on Molecular Assembly and Structural Stability of Bacteriorhodopsin Reconstituted into Phosphatidylcholine Liposomes with Different Acyl-Chain Lengths Yasunori Yokoyama,†,‡ Lumi Negishi,†,§ Taku Kitoh,† Masashi Sonoyama,†,| Yasuo Asami,⊥ and Shigeki Mitaku*,†,‡ Department of Applied Physics, Graduate School of Engineering, Nagoya UniVersity, Nagoya, 464-8603 Japan, and TA Instruments Japan, Inc., Tokyo, 141-0031 Japan ReceiVed: August 24, 2010; ReVised Manuscript ReceiVed: September 24, 2010

Previous studies on the correlation between bacteriorhodopsin (bR) disassembly and photobleaching suggested that a weakening of intermolecular interactions is responsible for irreversible photobleaching (Mukai, Y.; Kamo, N.; Mitaku, S. Protein Eng. 1999, 12, 755-759; Yokoyama, Y.; Sonoyama, M.; Mitaku, S. J. Biochem. 2002, 131, 785-790). In order to reveal the role of the lipid matrix in bR assembly and photobleaching, we reconstituted bR into diacylphosphatidylcholine (diacylPC) vesicles with three different saturated acyl-chain lengths. Visible circular dichroism (CD) spectra collected upon photobleaching showed an exciton-to-positive transition for bR reconstituted into dimyristoyl-, dipalmitoyl-, and distearoyl-PC vesicles around 17, 35, and 50 °C, respectively. These transition temperatures were close to the main transition temperature of reconstituted vesicles measured by calorimetry, indicating that the lipid phase transition brought about protein disaggregation. Absorption spectra of reconstituted bR exhibited a blue-shifted retinal absorption during protein disaggregation in the ground state. Absorption spectra collected from samples exposed to continuous illumination revealed an accumulation of M-intermediate state, and the absorption band around 410 nm underwent a blue shift through the visible CD change, indicating conformational perturbations due to protein disassembly. Irreversible photobleaching started to occur at the same temperature range as the change in the visible CD spectrum, clarifying the correlation between bR disassembly and photobleaching. In contrast, no thermal bleaching was observed below 60 °C for any sample kept in the dark. A plausible model for irreversible photobleaching is presented, on the basis of these experimental results. I. Introduction Bacteriorhodopsin (bR), originally identified in the cytoplasmic membrane of Halobacterium salinarum, is a light-driven proton pump.1 It is known that bR molecules spontaneously assemble with each other in the plasma membrane, forming membrane patches consisting of a two-dimensional (2D) crystalline array of bR commonly called purple membrane (PM).2 Proton translocation across this membrane involves several spectrally distinguishable photointermediate states (J, K, L, M, N, and O) of the photocycle which are triggered upon absorption of visible light by retinal chromophores covalently bound to bR via a Schiff base linkage.3 Comparisons of the molecular structures of the photointermediate states and the ground state have shown that the opening movements of the cytoplasmic proton channel during the M- and N-intermediate states are essential for unidirectional proton translocation.4-6 * Corresponding author. E-mail: [email protected]. Phone: +81-52-789-4466. Fax: +81-52-789-3706. † Department of Applied Physics, Graduate School of Engineering, Nagoya University. ‡ Present address: Department of Computational Science and Engineering, Graduate School of Engineering, Nagoya University, Nagoya, 464-8603 Japan. § Present address: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501 Japan. | Present address: Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Kiryu, 376-8515 Japan. ⊥ TA Instruments Japan, Inc.

Irreversible photobleaching phenomenon, which is considered to be inappropriate for its original role as a light-driven proton pump, has recently been reported to occur when bR molecules were irradiated with visible light, even in the absence of hydrolysis reagents such as hydroxylamine.7-14 That this phenomenon occurs in a light intensity-dependent manner8 and causes irreversible conformational changes in the bR polypeptide chain15 strongly suggests that irreversible photobleaching is a kind of protein denaturation induced by a photoreaction of bR. Irreversible photobleaching of bR was observed at high temperature and high pH in PM,7-11 and has been reported to occur even at room temperature in bR solubilized with mild nonionic detergents such as octyl-β-glucoside and Triton X-100.12,13 These results strongly suggest that intermolecular interactions between bR molecules in the 2D-crystalline array are essential for structural recovery from the photoexcited states. An experimental system involving reconstitution of bR into phospholipid bilayer vesicles with phosphatidylcholine (PC) headgroups would be useful for clarifying the nature of irreversible photobleaching, because lipid phase transition from the gel (Lβ′) phase to the liquid-crystalline (LR) phase was shown to induce disassembly of the 2D-crystalline packing of bR when the protein was reconstituted into a dimyristoylphosphatidylcholine (DMPC) bilayer.16,17 Moreover, time-resolved absorption spectroscopy of bR reconstituted into a DMPC bilayer showed that a spectrum of the late M-intermediate state underwent a blue shift of approximately 15 nm in the LR phase, whereas two decay-time substates of the M-state were observed at about

10.1021/jp108034n  2010 American Chemical Society Published on Web 11/08/2010

Assembly of bR Reconstituted into DiacylPC Vesicles the same maximum wavelength in the Lβ′ phase.18 This result strongly suggested that disassembly of bR in the LR phase leads to structural perturbation around the retinal chromophore in the late M-state, which is responsible for the conformational switch from proton release to proton uptake during the photocycle.3 Because the lipid phase transition temperature depends on acylchain length as well as the unsaturated bond, an experiment using diacylPC bilayer vesicles with various lengths of the saturated acyl-chains should provide information about correlation between the intermolecular interaction and thermal effects upon the molecular structure and structural stability. In this work, we examined photobleaching in a reconstituted system using diacylPC bilayer vesicles with three different acylchain lengths. Visible circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) were employed for monitoring the bR assembly state and lipid phase transition, respectively. We also investigated the molecular structural changes occurring mainly adjacent to the retinal chromophore, both in the dark and under illumination with visible light. On the basis of our experimental results, we propose a plausible model describing the effect of lipid phase transition upon bR assembly state, molecular structure, and structural stability. II. Materials and Methods Sample Preparation. Three phospholipids with PC headgroups, specifically dimyristoylphosphatidylcholine (DMPC; diC14:0 PC), dipalmitoylphosphatidylcholine (DPPC; diC16:0 PC), and distearoylphosphatidylcholine (DSPC; diC18:0 PC), were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Triton X-100 and other chemical reagents for buffer solutions were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Purple membranes (PM) of H. salinarum, strain R1M1, were purified according to standard procedures,19 and then resuspended in 25 mM phosphate buffer (pH 7.2). bR was reconstituted into diacylPC bilayer vesicles according to the following method. PM (bR concentration of 20 µM) were solubilized with 10 mM Triton X-100 at 25 °C for 12 h in the dark. Solubilized bR was contained in the supernatant after ultracentrifugation at 105 000 g for 1 h. Solubilized bR was mixed with a liposome suspension at a molar ratio of 1:150, and then the mixture was incubated for 5 h at 25 °C in the dark. Detergents were subsequently removed by addition of BioBeads SM-2 polystyrene beads (Bio-Rad Laboratories, Inc., Hercules, CA) and incubation for 17 h at 27 °C in the dark with gentle stirring. After removal of the Bio-Beads, reconstituted bR was suspended in 25 mM phosphate buffer (pH 7.2). The final concentrations of bR and lipid for the bilayer vesicles were approximately 7 µM and 1.5 mM, respectively. Spectroscopy. Absorption and visible CD spectra of bR reconstituted into bilayer vesicles were recorded on an Agilent 8453 spectrophotometer equipped with an Agilent 89090A Peltier temperature controller (Agilent Technologies, Santa Clara, CA), and a J-820 spectropolarimeter equipped with a JWJTC-484 Peltier temperature controller (Jasco Co., Tokyo, Japan), respectively. Absorption and visible CD spectra were collected at a temperature range 5-80 °C. Each CD spectrum was recorded at a scan rate of 100 nm min-1 and a data acquisition time of 1 s, and 8 scans were averaged. A fast Fourier transform (FFT) algorithm was used to reduce the noise of CD spectra. For absorption measurements collected under illumination with continuous visible light, a previously described experimental setup9,10 was combined with the spectrophotometer. A baseline correction for absorption spectra was performed to

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Figure 1. Temperature-dependent CD spectral changes in the visible region for bR reconstituted into diacylPC vesicles. (a) Visible CD spectra of bR reconstituted into DPPC vesicles measured below and above the lipid phase transition temperature. Thin and thick lines represent the spectra before and after smoothing with a FFT algorithm, respectively. (b) Temperature dependence of molar ellipticity at 600 nm after smoothing for bR reconstituted into (i) DMPC, (ii) DPPC, and (iii) DSPC vesicles. Arrows represent the midpoint of the visible CD change from the exciton- to the positive-type spectrum.

eliminate light scattering by subtracting the power function of the wavelength.9 Calorimetry. Calorimetric analysis of bR reconstituted into bilayer vesicles was carried out using a Nano DSC differential scanning calorimeter equipped with 0.3 mL platinum capillary cells (TA Instruments, Inc., New Castle, DE). The DSC scans were recorded across a temperature range 5-60 °C with a scan rate of 1 °C min-1. The concentrations of bR and lipids in the calorimetric measurements were approximately 7 µM and 1.5 mM, respectively. Temperature-Jump Experiment. In order to estimate the degree of photobleaching and thermal bleaching, a previously described temperature-jump experiment9,10 was conducted involving bR reconstituted into bilayer vesicles. Briefly, 800 µL of a stock solution of reconstituted vesicles ([bR] ≈ 6-7 µM) was added to 1600 µL of preheated buffer solution in a cuvette to raise the sample temperature. The sample temperature was maintained during a 1 h incubation with or without continuous visible light illumination. After the incubation, samples were rapidly cooled down and stored overnight in a refrigerator with protection from light. Absorption spectra of thermal bleached or photobleached bR samples were collected at room temperature in the dark the next day. The degree of bleaching which occurred during the incubation at high temperature was estimated by dividing the absorbance at 560 nm after cooling by that obtained prior to the temperature-jump. III. Results and Discussion Figure 1a shows representative visible CD spectra of bR reconstituted into DPPC bilayer vesicles at temperatures below and above the transition point from the Lβ′ to the LR phase (42 °C). The visible CD spectrum of the Lβ′ phase exhibited an asymmetric exciton-type CD band centered at 560 nm, whereas a positive-type CD band was observed in CD spectra of the LR phase, consistent with previous reports.16,18 This CD spectral change indicates that molecular disassembly to the monomeric

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Figure 2. Excess heat capacity curves for bR reconstituted into diacylPC vesicles: (i) DMPC, (ii) DPPC, and (iii) DSPC. Arrows represent the midpoint of the visible CD change.

state16 occurred during the lipid phase transition to the LR phase. The same visible CD spectral change was observed with bR reconstituted into DMPC and DSPC vesicles. The CD intensity at 600 nm (i.e., the center of the negative lobe of the excitontype CD band) was used to analyze the exciton-to-positive transition in the visible CD spectrum. The CD intensity at 600 nm of bR reconstituted into bilayer vesicles was temperaturedependent (Figure 1b). As shown in Figure 1b, the midpoints of the visible CD changes were approximately 17, 35, and 50 °C for DMPC-bR, DPPC-bR, and DSPC-bR, respectively. The DSC profiles of bilayer vesicles reconstituted with bR are shown in Figure 2. The incorporation of bR into DMPC vesicles brought about a downshifting and broadening of the main transition peak with a second component, possibly due to the influence of lipid heterogeneity on protein-lipid interactions.17 The main transition peaks were observed at approximately 20, 38, and 53 °C for DMPC-bR, DPPC-bR, and DSPC-bR, respectively. These transition temperatures were close to the midpoints of the visible CD changes for the three variable chain-length reconstituted bilayer vesicles, strongly suggesting that lipid phase transition from the Lβ′ to the LR phase led to bR disassembly. A second peak was observed for DPPC-bR and DSPC-bR vesicles at approximately 24 and 30 °C, respectively (Figure 2). The origin of the second peak is discussed below. To investigate the effect of protein disassembly induced by the lipid phase transition upon the molecular structure of bR, we monitored retinal absorption in the dark of bR reconstituted into bilayer vesicles. It is generally held that retinal absorption reflects retinal conformation as well as the structures around the retinal pocket.20 Figure 3a shows representative UV-vis absorption spectra of bR reconstituted into DPPC vesicles at various temperatures (upper column), and the spectral difference from a spectrum measured at the lowest temperature during the experiments (10 °C) (lower column). The absorption spectra exhibited a gradual blue shift from 560 nm below 20 °C to 530 nm at 60 °C with decreasing absorption intensity. The difference spectra show that the absorption at 570 nm decreased with increasing temperature, concomitant with an absorption increase at 460 nm. This spectral change, which was observed for the three differing acyl-chain length reconstituted diacylPC vesicles, was quite similar to the change reported for bR in PM at temperatures above 60 °C.10,21 Figure 3b shows the temperature dependence of retinal absorption changes at 460 and 570 nm for the reconstituted bilayer vesicles. The retinal absorption changes were accelerated around the midpoints of the visible CD change in the three types of reconstituted vesicles. This result clearly indicates that the molecular structure of bR reconstituted into bilayer vesicles is considerably altered by protein disassembly due to the lipid phase transition, mainly

Figure 3. Temperature-dependent retinal absorption change of bR reconstituted into diacylPC vesicles in the dark. (a) Absorption spectra of bR reconstituted into DPPC vesicles recorded at the indicated temperature (upper column) and spectral differences from the spectrum collected at the lowest temperature (lower column). (b) Temperature dependence of absorption changes at 460 nm (O) and 570 nm (b) for bR reconstituted into (i) DMPC, (ii) DPPC, and (iii) DSPC vesicles. Arrows indicate the midpoint of the visible CD change for the three differing acyl-chain length reconstituted vesicles.

Figure 4. Temperature-dependent retinal absorption change of bR reconstituted into diacylPC vesicles under illumination with visible light. (a) Absorption spectra of bR reconstituted into DMPC vesicles recorded at the indicated temperature. (b) Temperature dependence of the maximum wavelength of the photoproduct around 410 nm (Mintermediate state) for bR reconstituted into (i) DMPC, (ii) DPPC, and (iii) DSPC vesicles. Arrows represent the midpoint of the visible CD change.

around the retinal pocket. In the case of bR reconstituted into DPPC and DSPC liposomes, however, slight absorption changes were observed at lower temperatures than their main transitions. UV-vis absorption spectra of bR reconstituted into DMPC vesicles under visible light illumination are shown in Figure 4a. This measurement was taken 5 min (just above the dead time of the temperature jump) after the temperature jump with visible light illumination to minimize the bleaching effect. Absorption at 410 nm was observed under visible light illumination, especially below 20 °C, while a slight absorption band was detected around 400 nm above 30 °C. This absorption band disappeared immediately when illumination was turned

Assembly of bR Reconstituted into DiacylPC Vesicles

Figure 5. (a) Absorption spectra of bR reconstituted into DPPC vesicles after rapid cooling following a 1 h incubation at the indicated temperature, in the dark (upper column) and under illumination (lower column). Broken line represents the spectrum before the temperature jump. (b) Temperature dependence of the degree of spectral recovery of the retinal absorption around 560 nm induced by high-temperature incubation in the dark (b) and under illumination (O) for bR reconstituted into DMPC (i), DPPC (ii), and DSPC (iii) vesicles. Arrows represent the midpoint of the visible CD change.

off. It was reported that the decay time of the M-intermediate state during the photocycle increases considerably in the reconstituted bilayer environment for both the Lβ′ and the LR phases.22 Therefore, we considered that the absorption observed around 410 nm results from the M-intermediate state, since our spectrophotometer, which is equipped with a photodiode array detector, can collect spectra at 0.1 s intervals. Figure 4b shows the temperature dependence of the maximum wavelength of the absorption band around 410 nm accumulated by light illumination for the three differing acyl-chain length reconstituted bilayer vesicles. A significant blue shift in the accumulated M-intermediate state was observed for each of the reconstituted vesicle types above the temperatures of the visible CD change. Time-resolved absorption spectroscopy of bR reconstituted into DMPC bilayer vesicles revealed a blue shift in spectra of the late M-intermediate state at the LR phase.18 It is believed that the late M-intermediate state is essential for switching from proton release to proton uptake during the photocycle.3 Our results strongly suggest that protein disassembly induced by the lipid phase transition affects not only the molecular structure in the dark, but also the functional intermediate state that is responsible for conformational switching under illumination. To elucidate the influence of protein disassembly upon the structural stability of bR reconstituted into bilayer vesicles, we investigated retinal absorption recovery in high-temperature incubations with and without light illumination. Figure 5a shows absorption spectra of bR reconstituted into DPPC vesicles and incubated for 1 h at high temperature in the dark and then cooled (upper), and the corresponding spectra incubated under visible light illumination (lower). At high temperature, the retinal absorption around 560 nm underwent a blue shift both in the dark and under illumination, as shown in Figures 3a and 4a. Upon cooling down the sample temperature, the maximum wavelength of retinal absorption completely recovered to the pre-temperature-jump level, whereas an unrecoverable fraction of absorption was observed under illumination. Figure 5b

J. Phys. Chem. B, Vol. 114, No. 47, 2010 15709 illustrates the temperature dependence of the recovered fraction of retinal absorption for the three types of reconstituted bilayer vesicles after cooling from the high-temperature incubation. For samples which were incubated in the dark, the retinal absorption completely recovered when the temperature fell below 60 °C, while a significant reduction of the reversible fraction was observed starting around the temperatures of the visible CD change for samples which were incubated with visible light illumination. These results clearly indicate that bR reconstituted into bilayer vesicles undergoes photobleaching when bR molecules are disassembled by the lipid phase transition. In the case of DPPC-bR and DSPC-bR liposomes, however, slight photobleaching was observed beginning at a temperature below the main transition point. The effects of lipid phase transition upon the molecular assembly, molecular structure, and structural stability of bR reconstituted into bilayer vesicles are summarized in Figure 6. In the Lβ′ phase, bR molecules in the bilayer vesicle are assembled with each other to form a nativelike assembly state. The nativelike retinal absorption in the Lβ′ phase underwent a blue shift, together with protein disassembly induced by the lipid phase transition to the LR phase. A blue shift of retinal absorption of the accumulated M-intermediate state and significant photobleaching were also observed during protein disassembly. These results imply that weakening of intermolecular interactions due to protein disassembly induced by the lipid phase transition influences the molecular structure and structural stability of bR reconstituted vesicles, especially under illumination. The retinal absorption change in the dark, which was characterized by increase at 460 nm and a decrease at 570 nm, was very similar to that reported for the high-temperature intermediate state of bR in PM.10 Since the helical conformational change from RII to RI helix, an increase of retinal 13-cis isomer, and an increase of water accessibility to the Schiff base were reported for this state which appeared when the 2Dcrystalline array started to disaggregate,23,24 it is likely that bR molecule assumes a more relaxed form in the LR phase at which intermolecular interactions weakened. Time-resolved absorption spectroscopy of bR reconstituted into DMPC bilayers revealed that the blue shift of the late M-intermediate state occurred at the point at which the visible CD changed from an exciton to a positive shape around the main transition.18 A blue shift in the late M-intermediate state of monomeric bR solubilized with Triton X-100 has also been reported.25 It is reasonable to conclude that the blue shift of the accumulated M-intermediate state we observed results from structural perturbation in the late M-intermediate state. Since the late M-intermediate state is believed to be essential for switching between the “closed” and “opened” states of the cytoplasmic proton channel during the photocycle,3 it is likely that the perturbation in this functional intermediate state rules out the ordinary structural recovery to the ground state and causes irreversible photobleaching. In this work, we observed a slight retinal absorption change in the dark as well as slight photobleaching of DPPC-bR and DSPC-bR at around 25 °C, even below their main transitions. The second peaks, at approximately 25 and 30 °C for DPPCbR and DSPC-bR, respectively, are shown in Figure 2. Although the origin of this DSC peak is still unknown, we attributed it to the pretransition of the bilayer vesicle from the gel (Lβ′) to the ripple (Pβ′) phases. The pretransition temperatures of the pure liposomes are higher (36 °C for DPPC and 51 °C for DSPC) than those of the reconstituted vesicles. However, it is likely that the pretransition temperature becomes lower since incorporation of acylated peptides into DPPC bilayers causes a

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Figure 6. Schematic model of effect of lipid phase transition upon molecular assembly, protein structure, and structural stability of bR reconstituted into diacylPC bilayer vesicles: (a) nativelike ground state, (b) nativelike M-intermediate state, (c) high-temperature intermediate-like ground state, and (d) blue-shifted M-intermediate state. CP side: cytoplasmic side. PIs: photointermediates.

gradual downshifting of the pretransition and the main transition temperatures with increasing peptide/lipid ratios.26 Solid-state 13 C NMR and electron spin resonance (ESR) spectroscopy indicated that the spectrum of DPPC in the Pβ′ phase could be reproduced by the superposition of two spectra taken at the Lβ′ and LR phases.27,28 ESR spectroscopy of the stearic acid spin probe revealed that approximately 20% of the total acyl chain in the Pβ′ phase assumes a disordered form similar to the LR phase, strongly suggesting that the Pβ′ phase possesses the structural features similar to both the Lβ′ and LR phases.28 Moreover, ultrasonic velocity measurements showed that the membrane softens beyond the pretransition.29 These experimental results suggest that partial structural change and photobleaching of bR could occur at temperatures around 30 °C at which point the physical properties of the bilayer changed. On the other hand, it is also likely that the second DSC peaks observed around 30 °C represent protein conformational changes. Kinetic measurements of bR reconstituted into DMPC vesicles demonstrated that the photobleaching rate constant drastically increases above temperatures around 30 °C (Negishi and Mitaku, unpublished results). It is possible that common structural characteristics of bR reconstituted into diacylPC vesicles that appear above 30 °C were observed in this work as well. The origin of the slight structural changes and photobleaching that occurred around 30 °C should be investigated further. IV. Conclusions We investigated the effect of molecular assembly of bR reconstituted into the DMPC, DPPC, and DSPC bilayer vesicles upon the molecular structure and structural stability of bR. Visible CD spectra showed that the exciton-to-positive transition resulting from protein disassembly of bR reconstituted into DMPC, DPPC, and DSPC vesicles occurs around 17, 35, and 50 °C, respectively. DSC profiles of the reconstituted bilayer vesicles indicated that the main transition temperatures are very close to the visible CD transition temperature, suggesting that the lipid phase transition to the LR phase brought about protein disaggregation. Absorption spectra of the reconstituted bilayer vesicles demonstrated the presence of conformational perturba-

tions in both the ground state and the M-intermediate state during protein disassembly. Irreversible photobleaching was observed at the same temperatures as the visible CD change, clarifying the relationship between bR disaggregation and irreversible photobleaching. We proposed a model for irreversible photobleaching as shown in Figure 6. It is likely that the conformational perturbations in the M-intermediate state, which were caused by protein disassembly due to the lipid phase transition to the LR phase, are responsible for irreversible photobleaching. Acknowledgment. We thank Dr. I. Hatta of the Japan Synchrotron Radiation Research Institute (JASRI/Spring-8) for helpful discussions about the lipid phase transition. This work was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Monbukagakusho) and by SENTAN, JST. References and Notes (1) Bogomolni, R. A.; Baker, R. A.; Lozier, R. H.; Stoeckenius, W. Light-driven proton translocations in Halobacterium halobium. Biochim. Biophys. Acta 1976, 440, 68–88. (2) Henderson, R. The purple membrane from Halobacterium halobium. Annu. ReV. Biophys. Bioeng. 1977, 6, 87–109. (3) Lanyi, J. K. Bacteriorhodopsin. Annu. ReV. Physiol. 2004, 66, 665– 688. (4) Henderson, R.; Baldwin, J. M.; Ceska, T. A.; Zemlin, F.; Beckmann, E.; Downing, K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 1990, 213, 899– 929. (5) Luecke, H.; Schobert, B.; Richter, H.-T.; Cartailler, J.-P.; Lanyi, J. K. Structure of bacteriorhodopsin at 1.55 Å resolution. J. Mol. Biol. 1999, 291, 899–911. (6) Lanyi, J. K.; Schobert, B. Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K, L, M1, M2, and M2′ intermediates of the photocycle. J. Mol. Biol. 2003, 328, 439– 450. (7) Etoh, A.; Itoh, H.; Mitaku, S. Light-induced denaturation of bacteriorhodopsin just above melting point of two-dimensional crystal. J. Phys. Soc. Jpn. 1997, 66, 975–978. (8) Dancsha´zy, Z.; Tokaji, Z.; De´r, A. Bleaching of bacteriorhodopsin by continuous light. FEBS Lett. 1999, 450, 154–157.

Assembly of bR Reconstituted into DiacylPC Vesicles (9) Yokoyama, Y.; Sonoyama, M.; Mitaku, S. Irreversible photobleaching of bacteriorhodopsin in a high-temperature intermediate state. J. Biochem. 2002, 131, 785–790. (10) Yokoyama, Y.; Sonoyama, M.; Mitaku, S. Inhomogeneous stability of bacteriorhodopsin in purple membrane against photobleaching at high temperature. Proteins 2004, 54, 442–454. (11) Yokoyama, Y.; Sonoyama, M.; Nakano, T.; Mitaku, S. Structural changes of bacteriorhodopsin in the purple membrane above pH 10 decreases heterogeneity of the irreversible photobleaching components. J. Biochem. 2007, 142, 325–333. (12) Mukai, Y.; Kamo, N.; Mitaku, S. Light-induced denaturation of bacteriorhodopsin solubilized by octyl-β-glucoside. Protein Eng. 1999, 12, 755–759. (13) Sasaki, T.; Sonoyama, M.; Demura, M.; Mitaku, S. Photobleaching of bacteriorhodopsin solubilized with Triton X-100. Photochem. Photobiol. 2005, 81, 1131–1137. (14) Sonoyama, M.; Fukumoto, M.; Kuwabata, Y. Highly stable solubilization of membrane protein bacteriorhodopsin with a short-chain phospholipid diheptanoylphosphatidylcholine. Chem. Lett. 2010, 39, 876– 877. (15) Yokoyama, Y.; Sonoyama, M.; Mitaku, S. Structural changes in bacteriorhodopsin in purple membranes induced by irreversible photobleaching with heterogeneous and homogeneous stability. Photochem. Photobiol. 2010, 86, 297–301. (16) Heyn, M. P.; Cherry, R. J.; Dencher, N. A. Lipid-protein interactions in bacteriorhodopsin-dimyristoylphosphatidylcholine vesicles. Biochemistry 1981, 20, 840–849. (17) Heyn, M. P.; Blume, A.; Rehorek, M.; Dencher, N. A. Calorimetric and fluorescence depolarization studies on the lipid phase transition of bacteriorhodopsin-dimyristoylphosphatidylcholine vesicles. Biochemistry 1981, 20, 7109–7115. (18) Sonoyama, M.; Kikukawa, T.; Yokoyama, Y.; Demura, M.; Kamo, N.; Mitaku, S. Effect of molecular assembly on photocycle of reconstituted bacteriorhodopsin: Significant blue shift of the late M photointermediate in the liquid crystalline phase. Chem. Lett. 2009, 38, 1134–1135.

J. Phys. Chem. B, Vol. 114, No. 47, 2010 15711 (19) Oesterhelt, D.; Stoeckenius, W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974, 31, 667–678. (20) Hayashi, S.; Tajkhorshid, E.; Pebay-Peyroula, E.; Royant, A.; Landau, E. M.; Navarro, J.; Schulten, K. Structural detarminants of spectral tuning in retinal proteinssbacteriorhodopsin vs sensory rodopsin II. J. Phys. Chem. B 2001, 105, 10124–10131. (21) Neebe, M.; Rhinow, D.; Schromczyk, N.; Hampp, N. A. Thermochromism of bacteriorhodopsin and its pH dependence. J. Phys. Chem. B 2008, 112, 6946–6951. (22) Dencher, N. A.; Kohl, K.-D.; Heyn, M. P. Photochemical cycle and light-dark adaptation of monomeric and aggregated bacteriorhodopsin in various lipid environments. Biochemistry 1983, 22, 1323–1334. (23) Wang, J.-P.; El-Sayed, M. A. The effect of protein conformation change from RII to RI on the bacteriorhodopsin photocycle. Biophys. J. 2000, 78, 2031–2036. (24) Sonoyama, M.; Mitaku, S. High-temperature intermediate state of bacteriorhodopsin prior to the premelting transition of purple membrane revealed by reactivity with hydrolysis reagent hydroxylamine. J. Phys. Chem. B 2004, 108, 19496–19500. (25) Va´ro´, G.; Lanyi, J. K. Effects of crystalline structure of purple membrane on the kinetics and energetics of the bacteriorhodopsin photocycle. Biochemistry 1991, 30, 7165–7171. (26) Pedersen, T. B.; Kaasgaard, T.; Jensen, M.Ø.; Frokjaer, S.; Mouritsen, O. G.; Jørgensen, K. Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides. Biophys. J. 2005, 89, 2494–2503. (27) Wittebort, R. J.; Schumidt, C. F.; Griffin, R. G. Solid-state carbon13 nuclear magnetic resonance of the lecithin gel to liquid-crystalline phase transition. Biochemistry 1981, 20, 4223–4228. (28) Tsuchida, K; Hatta, I. ESR studies on the ripple phase in multilamellar phospholipid bilayers. Biochim. Biophys. Acta 1988, 945, 73– 80. (29) Aruga, S.; Kataoka, R.; Mitaku, S. Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes: I. Transition anomalies of ultrasonic properties. Biophys. Chem. 1985, 21, 265–275.

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