Langmuir 2000, 16, 5503-5505
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Long-Lived M-State in Multilayer Films Fabricated by Alternative Deposition of a Polycation and Bacteriorhodopsin Meiling Li, Baofang Li,* and Long Jiang Laboratory of Colloid and Interface, Center of Molecular Science, Institute of Chemistry, The Chinese Academy of Sciences, Beijing, P. R. China Tapio Tussila, Nikolai Tkachenko, and Helge Lemmetyinen Institute of Materials Chemistry, Tampere University of Technology, FIN-33101 Tampere, Finland Received October 13, 1999. In Final Form: March 22, 2000
Introduction Bacteriorhodopsin (bR) is a photochromic protein present in the purple membrane (PM) isolated from Halobacterium salinarium.1 Its function is to absorb light and transfer this energy to the membrane potential. Upon absorption of light energy, bR undergoes a complex photocycle that involves several intermediates, namely K, L, M, N, and O, before reverting to the ground state (B-state).2 Of these intermediates in the photocycle, the M-state is the only one that is deprotonated and it is the most stable state with a lifetime of the order of 10 ms for aqueous native bR solution. As a result of deprotonation, the absorption wavelength is significantly blue-shifted from 570 nm for the B-sate to 412 nm for the M-state. It is this photochromism that provides a mechanism for optical device applications such as spatial light modulation3 and realtime holographic image processing.4 It is also known that if the lifetime of the M-state can be extended, then there would be a potential for roomtemperature optical storage application.5,6 Generally, water-soluble polymers such as poly(vinyl alcohol) and gelatin are used as a matrix to form thin solid bR-polymer films. In these films, bR molecules are incorporated into the matrix uniformly. Recently, a new and simple method for the preparation of thin heterogeneous bR multilayer films has been developed by He et al.7 using the alternate layer-by-layer electrostatic deposition of polyelectrolyte poly(dimethyldiallyammonium chloride) (PDAC) and bR. More recently, the photocurrent generated from this type of film has been further studied by the authors.8 However, the photochromic property of the M-state in such a film has not been reported so far. In the present study, a (PDAC/bR)90 multilayer film with an optical density (OD) of 0.30 at 560 nm has been * Corresponding author. Present address: Institute of Photographic Chemistry, The Chinese Academy of Sciences, De Wai Bei Sha Tan, Beijing 100101, P. R. China. Telephone: +86-10-64888175. Fax: +86-10-64879375. E-mail:
[email protected]. (1) Oesterhelt, D.; Stoeckenius, W. Nat. New Biol. 1971, 233, 149. (2) Lozier, R. H.; Bogomolni, R. A.; Stoeckenius, W. Biophy. J. 1975, 15, 955. (3) Thoma, R.; Hampp, N.; Brauchle, C. Opt. Lett. 1991, 16, 651. (4) Hampp, N.; Brauchle, C.; Oesterhelt, D. Boiphys. J. 1990, 58, 83. (5) Chen, Z.; Lewis, A.; Takei, H.; Nebenzahl, I. Appl. Opt. 1991, 30, 5188. (6) Wang, C.; Bacon, M.; Kar, A. K.; Wherrett, B. S.; Baxter, R. L. Jpn. Appl. Phys. 1997, 36, 439. (7) He, J.-A.; Samuelson, L.; Li, L.; Kumar, J.; Tripathy, S. K. Langmuir. 1998, 14, 1674. (8) He J-A,; Samuelson, L.; Li L.; Kumar, J.; Tripathy, S. K. J. Phys. Chem. B 1998, 102, 7067.
Figure 1. Absorption spectra of PDAC/bR multilayers. From bottom to top, the curves correspond to 10, 20, 30, 40, 50, 60, 70, 80, and 90 bilayers, respectively. The inset shows a linear increase of OD at 560 nm with the deposition number.
successfully formed and the decay kinetics of the M-state in the film have been studied by both spectrophotometry and flash photolysis. Materials and Methods Purple membrane was isolated from Halobacterium salinarium using the procedures described by Oesterhelt and Stoeckenius.9 The concentration of bR in 50 mM tris(tris(hydroxymethyl)aminomethane) buffer solution at pH 8 was 0.5 mg/mL before dipping. The concentration of poly(dimethyldiallylammonium chloride) polyelectrolyte was 2 mg/mL in 0.5 M NaCl buffer solution (KH2PO4/K2HPO4) at pH 6.8. The preparation of the (PDAC/bR)90 multilayer on a quartz plate was done according to ref 5. A quartz plate with a negatively charged surface was immersed in 2 mg/mL PDAC solution for 5 min and then rinsed with Mili-Q water for 2 min. After being dried under nitrogen, the layered support was immersed in bR suspension for 5 min and dried again under nitrogen. This process was repeated until (PDAC/bR)90 multilayer was formed. The absorption measurements of the film were carried out with a UV-250IPC spectrophotometer. A frequency-doubled Nd:YAG laser at a wavelength of 532 nm was used as the exciting light source. A halogen lamp was used as the monitoring light source. To increase the signal-to-noise ratio, the optical signal was averaged 10-30 times. Several wavelengths corresponding to the absorption of the intermediates were monitored. All the measurements were performed at room temperature and at a relative humidity of 35%.
Results and Discussion The absorption spectra of the (PDAC/bR)n multilayers after each deposition of 10 bR layers are presented in Figure 1. The absorption maximum of the light-adapted form of the (PDAC/bR)90 multilayer is located at 560 nm, and the profile of this spectrum is the same as that of native bR in suspension, indicating that the physiological activity of bR in the multilayers fabricated by electrostatic deposition is preserved. Only a slight shift of 10 nm occurs compared to that of native bR in suspension. This blue shift is due to the influence of the dehydration on the retinal Schiff base of bR in a dry film.10 In addition, from (9) Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, 31, 667. (10) Hildebrant, P.; Stockburger, M. Biochemistry 1984, 23, 5539.
10.1021/la991344+ CCC: $19.00 © 2000 American Chemical Society Published on Web 05/19/2000
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Figure 2. Thermal reversion of the M-state to the B-state in (PDAC/bR)90 multilayer film after 30 s continuous-wave photoexcitation (>530 nm).
Figure 3. Decay kinetics of the M-state and recovery kinetics of the B-state in (PDAC/bR)90 film following excitation by a laser pulse at 530 nm.
the inset of Figure 1, it can be seen that the absorbency of bR at 560 nm is a linear function of the number of bilayers deposited, suggesting that the reproducibility of the technique for deposition number 1-90 is quite good. We therefore conclude that the layer-by-layer deposition of bR in the (PDAC/bR)90 multilayer occurs rather uniformly. The thermal relaxation from the M-state to the B-state for the (PDAC/bR)90 film is shown in Figure 2. The subsequent absorption spectra were obtained as the M-state relaxed back to the B-state after 30 s continuouswave photoexcitation (>530 nm, selected by a cutoff filter). Owing to the limitation of the instrument, the spectrum at the beginning of the decay of the M-state was obtained indirectly from the time dependence of absorbency monitored at different wavelengths. From this figure, it can be seen that ∼80% of the M-state decays into the B-state in ∼15 min and all of the M-state decays into the B-state in ∼30 min. Thus, the M-state lifetime in (PDAC/bR)90 film is estimated to be about 15 min, in accordance with the reports by other authors using the same method.11 This long M-state lifetime represents a magnitude improvement of 4 orders over the 10 ms of the native bR protein and provides significant potential for optical application even under ambient conditions. To understand the mechanism of the M-state and its relaxation to the B-state, the photodynamics of the (PDAC/ bR)90 film were studied at several wavelengths, corresponding to the absorption of the intermediates, by laser flash photolysis. The 30 ns pulses at 532 nm, generated by a Nd:YAG laser, were used to pump the bR molecule through a single photocycle. Figure 3 shows two typical kinetic curves monitored at 410 and 560 nm, respectively. The decay kinetics of the M-state are analyzed in terms of a four-exponential function of A ) A1e-t/τ1 + A2e-t/τ2 + A3e-t/τ3 + A4e-t/τ4 and four time constants, 10 ms, 350 ms, 20 s, and 200 s, are obtained. The maximum time scale of the instrument is 500 s, and at least four time constants exist during the whole decay process of the M-state. The decay rate of the M-state at 410 nm is almost the same as the recovery rate of the B-state at 560 nm, except for the fast rise within 0-100 ms at 560 nm. This fast rise is probably a result of the fast decay of the K-like intermediate to the B-state at ∼600 nm with a time
constant of 10 ms (The K-state in the trans-photocycle decays normally to the L-state in 3 µs).12 Kinetics assignable to the O-state are not observed in the (PDAC/ bR)90 multilayer films, possibly due to the direct decay of the M-state to the B-state. This is in accord with the report of the other types of bR film.13 Thus, it appears that, under ambient conditions, the M-state in (PDAC/bR)90 film relaxes directly to the B-state according to multiexponential kinetics. This multiplicity of the decay of the M-state has also been observed in other dried bR films,14,15 and generally it is suggested to originate from the dried protein, which has reduced flexibility and is fixed in different conformations. Consequently the dried bR sample is inhomogeneous, and the protons can return via several pathways, which leads to the multiexponential kinetics for the decay of the M-state. It has been reported that at least two steps are included in the decay of the M-statesfirst, isomerization of 13-cis to all-trans followed by reprotonation of the M-state. The decay of the M-state in (PDAC/bR)90 film is much slower than that in bR-soyaphosphatidylcholine LangmuirBlodgett(LB) film,16 which exhibits a biexponential decay with time constants of 150 ms and 3 s. Both films are formed uniformly layer-by-layer, and the main difference between the LB films and those used in the present study is the presence of PDAC, implying that PDAC in (PDAC/ bR)90 plays a key role in the prolongation of the M-state lifetime. First, the optical density of the PDAC/bR multilayer is higher than that of LB film containing the same number of bR layers. Therefore, the PDAC/bR layerby-layer assembly is more compact and denser than the corresponding LB film. Possibly, the resulting hindrance to bR molecules leads to a larger activation free energy for isomerization from 13-cis to all-trans in (PDAC/bR)90 film compared to that in LB film. Second, PDAC is positively charged at pH 6.8 and is able to interact favorably with the cytoplasmic side of bR, which presents a higher negative charge than its extrocellular side in
(11) Gross, R. B.; Izgi, K.; Birge, R. R. SPIE 1992, 1662, 186.
(12) Ganea, C.; Gergely, C.; Ludmann, K.; Varo, G. Biophys. J. 1997, 73, 2718. (13) Varo, G.; Keszthelyi, L. Biophys. J. 1983, 43, 47. (14) Korenstein, R.; Hess, B. Nature 1977, 270, 184. (15) Varo, G.; Keszthelyi, L. Eur. J. Biochem. 1987, 14, 163. (16) Lemmetyinen, H.; Ikonen, M. Trends Photochem. Photobiol. 1994, 3, 413.
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alkaline PM suspension.17,18 This interaction is similar to that between argine and an amino acid residue of bR with side chains that are negatively charged at pH 9, such as aspartic acid, glutamic acid, or tyrosine, 19 and may significantly decrease the rate of reprotonation of Schiff (17) Fisher, K. A.; Yanagimoto, K.; Stoeckenius, W. J. Cell Biol. 1978, 77, 611. (18) Keszthelyi, L.; Ormos, P. FEBS Lett. 1980, 109, 189. (19) Nakasako, M.; Kataoka, M.; Tokunaga, F. FEBS Lett. 1989, 254, 211.
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base. The investigation of the detailed mechanism of the slow decay of the M-state in (PDAC/bR)90 film is continuing. Acknowledgment. This work was supported by National Natural Science Foundation of China (Contract No. 69731010) and The Chinese Academy of Sciences (Contract No. KJ-951-A1-501-05). LA991344+
