Thermochromism of Bacteriorhodopsin and Its pH Dependence

spectral transitions were found, involving spectral states absorbing at 460, 519, and 630 nm. These thermochromic absorption changes were analyzed in ...
0 downloads 0 Views 209KB Size
6946

J. Phys. Chem. B 2008, 112, 6946–6951

Thermochromism of Bacteriorhodopsin and Its pH Dependence Martin Neebe, Daniel Rhinow, Nina Schromczyk, and Norbert A. Hampp* Department of Chemistry, UniVersity of Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany, and Material Sciences Center, D-35032 Marburg, Germany ReceiVed: NoVember 23, 2007; ReVised Manuscript ReceiVed: March 31, 2008

Purple membranes (PMs), which consist of the photochromic membrane protein bacteriorhodopsin (BR) and lipids only, show complex thermochromic properties. Three different types of reversible temperature-dependent spectral transitions were found, involving spectral states absorbing at 460, 519, and 630 nm. These thermochromic absorption changes were analyzed in the range from 10 to 80 °C. In dependence on the bulk pH value, hypsochromic or bathochromic shifts in the BR absorption spectra are observed in BR gels as well as in BR films. The thermochromic changes between both purple and blue or purple and red were quantified in the CIE color system. The molecular changes causing these effects are discussed, and a model is presented in terms of intramolecular protonation equilibriums. The thermochromic properties of BR may be of interest in applications like security tags, as this feature may complement the wellknown photochromic properties of BR. 1. Introduction Bacteriorhodopsin (BR), the only protein in the purple membrane (PM) of Halobacterium salinarum,1,2 is an astonishingly robust protein when assembled in its native lipid environment. Cryoelectron microscopy as well as X-ray diffraction studies have provided valuable insights into the structure and function of BR,3–8 and have therefore smoothened the progress of its technical applications. BR exhibits photochromic properties; i.e., color changes are induced by reversible light-mediated switching between various metastable spectroscopic states.9 Because of its chemical robustness and thermal stability, PM bears an enormous potential for technological applications like data storage and optical devices.10,11 In this study, we report about the thermochromic properties of BR which provide an additional pathway for reversible switching between colored states. We find that BR-containing samples display temperature-dependent hypsochromic as well as bathochromic shifts which are further controlled by the bulk pH, and are strongly affected by charged residues of PM. The pH-dependent thermochromism of BR is visible at physiological temperatures, and was analyzed by visual absorption as well as reflectance spectroscopy. The combination of the photochromic and thermochromic properties of BR as well as its high stability in a variety of preparations makes BR a promising multifunctional pigment for several technical applications. 2. Materials and Methods Suspensions of PM containing wild-type BR (BR-WT) and the mutant BR-E194Q, where glutamic acid in position 194 is exchanged for glutamine, were a gift from Dieter Oesterhelt’s group at the Max-Planck Institute of Biochemistry, Martinsried, Germany. PM was purified by standard procedures.12 Briefly, PM was purified by sucrose-density gradient centrifugation (D(+)-sucrose, biochemical grade, Acros Organics, Geel, Belgium) and washed three times with 1.5 mM phosphate buffer * Corresponding author. Phone: +49 6421 2825775. Fax: +49 6421 2825798. E-mail: [email protected].

(pH 6.8, Na2HPO4/KH2PO4, p.a. grade, Fluka Chemie, Buchs, Switzerland). Acetylated PM (Ac-BR) was prepared according to a published procedure.13 A total of 5 mL of a 64 µM PM suspension was mixed with an equal volume of saturated sodium-acetate solution. A total of 20 µL of acetic anhydride was added in two portions under constant stirring at 0 °C. Then the reaction mixture was titrated to pH 7.5-8.0 with 1 M NaOH. The reaction was stopped after 80 min by dilution with 30 mL of water. The membranes were washed three times with 40 mL of deionized water by centrifugation. For absorption spectroscopy, PM was incorporated in polyacrylamide (PAA) gels in order to avoid aggregation at low pH. For this purpose, 2 mL of membrane suspension, 2218 mL of 40% (w/v) acrylamide/bisacrylamide 29:1 (Genaxxon Bioscience, Bieberach, Germany), 5 µL of N,N,N′,N′-tetramethylendiamine (TEMED) (Genaxxon Bioscience, Bieberach, Germany), and 35 µL of 10% (w/v) ammoniumpersulfate (AMPS) (Genaxxon Bioscience, Bieberach, Germany) were mixed and layered between glass plates. After curing, the glass plates were removed and the PM/PAA gels were ready to use. The pH of the gels was adjusted by incubation in 0.1 M buffer solution containing 75 mM KCl for 12 h at 4 °C. The following buffers were used: phosphate buffer pH 6.9/5.9 (Na2HPO4/ KH2PO4), acetate buffer pH 4.9/4.5/4.2/3.5 (acetic acid/sodium acetate), and citrate buffer pH 2.9/2.2/1.6 (sodium citrate/HCl). Temperature-dependent absorption spectra were recorded on an UVIKON 922 spectrometer (Kontron), equipped with a thermostatted stage. BR-WT/PAA gels were sealed in cuvettes which were filled with the appropriate buffer. For reflectance spectroscopy, BR/gelatine films on glass were produced from aqueous gelatine solutions (1.0% w/v). Films of OD ) 2.8 at 560 nm were obtained. Films were acidified by titration with carnitine hydrochloride solution (0.1 M) and then equilibrated to 60% relative humidity at 20 °C. In order to keep the films at a constant humidity during measurement, they were sealed between glass plates. Reflectance spectra were recorded by an Avantes AVS-S2000 fiberoptic spectrometer using Navitar TV-zoom optics at 90°

10.1021/jp7111389 CCC: $40.75  2008 American Chemical Society Published on Web 05/21/2008

Thermochromism of Bacteriorhodopsin

J. Phys. Chem. B, Vol. 112, No. 23, 2008 6947

Figure 1. Thermochromism of BR-WT/PAA gels. PAA gels are placed on two adjacent Peltier elements which are set to two different temperatures. In each case, the element to the left is set to 45 °C and the element to the right is set to 10 °C. (A) BR-WT/PAA gel with pH 4.2: The color shifts from purple to blue with increasing temperature. (B) BR-WT/PAA gel with pH 6.9: The color shifts from purple to red with increasing temperature.

viewing angle and 45° illumination angle. Quantitative analysis of reflectance spectra was done, using the CIELAB color system, provided by the Commision Internationale de l’ E´clairage (CIE). The procedure and the table of standard values were taken from the standard practice of the American Society for Testing and Materials (ASTM designation E308-01).14 From spectra taken in 20 nm steps from 360 to 780 nm reflectance of the samples, the CIE tristimulus values XYZ for the CIE 1931, 2°-standard observer were computed. 3. Results and Discussion 3.1. pH-Dependent Thermochromism of BR. The temperature-dependent color shift can be demonstrated easily by placing a BR-WT/PAA gel onto two adjacent Peltier elements which are adjusted to two different temperatures, as shown in Figure 1. We observed a pH-dependent thermochromic behavior of BR. BR-WT/PAA gels set to neutral pH (pH 6.9) display a distinguished color shift from the purple color, observed at 10 °C, to the red upon heating to 45 °C (Figure 1B). An identical gel, set to pH 4.2, i.e., slightly acidic, appears purple at 10 °C but shifts to the blue at 45 °C (Figure 1A). The thermochromic color shifts are fully reversible. When BR-WT, embedded into PAA gels, is brought to even lower pH values (pH < 2), it appears blue, due to the formation of blue membrane. These gels show a color shift in the opposite direction upon heating; i.e., the color shifts from blue to purple (not shown). The thermochromism of BR/PAA films was analyzed by temperature-dependent UV-vis spectroscopy. We observed three different types of BR thermochromism, depending on the pH. In Figure 2, the temperature-dependent absorption spectra as well as the difference spectra of dark-adapted BR-WT/PAA gels, set to three distinct pH values (pH 6.9, pH 4.2, and pH 2.2), are shown. At neutral pH (pH 6.9), the absorption band of dark-adapted BR-WT at 562 nm decreases upon heating, and a hypsochromic shifted species arises (Figure 2A). This behavior was named the type-1 transition. In the difference spectrum, a decrease at 577 nm and an increase at 460 nm are observed with an isosbestic point at 515 nm. Lowering the pH value of the BR-WT/PAA gels slightly from pH 6.9 to pH 4.2 completely changes the temperature-dependent spectral behavior. Now, a bathochromic shift of the absorption band is observed upon heating (Figure 2C). In the difference spectra, an increase in absorption at 635 nm and a decrease at 554 nm is observed with an isosbestic point at 573 nm (Figure 2D). This behavior was named the type-2 transition.

Figure 2. Absorption spectra (graphs A, C, and E) and the related difference spectra (graphs B, D, and F) of the different pH-dependent as well as temperature-dependent spectral transitions of dark-adapted BR-WT/PAA gels. (A and B) Type-1 transition, observed at pH 6.9, which goes along with a reversible hypsochromic shift upon heating to 77 °C. The decrease at 577 nm is accompanied by a rise in 460 nm absorption. (C and D) Type-2 transition, observed at pH 4.2, showing a reversible bathochromic shift up to 64 °C with a decrease in 560 nm absorption, accompanied by a rise in 630 nm absorption. (E and F) Type-3 transition, observed at pH 2.2, showing a reversible hypsochromic shift up to 45 °C, accompanied by a decrease in 628 nm and a rise in 519 nm absorption. An isosbestic point is found at 572 nm. At higher temperatures, BR denatures irreversibly and the known 380 nm absorption of free retinal arises (dashed lines). In each case, the lowest temperature was used as a reference for the calculation of the difference spectra.

Finally, a third type of transition is observed for BR-WT at pH 2.2 or lower. In this case, a temperature-dependent decrease in absorption at 630 nm and a concomitant increase of a state absorbing around 520 nm is observed (Figure 2E and F). In all types of spectral transition, we find an isosbestic point, indicating that two-state equilibriums are involved. The spectral changes are reversible within a range which depends on the sample. Above a certain threshold temperature, thermal degradation of the material takes place, which is indicated by dashed lines in the spectra (Figure 2). This threshold temperature decreases with lowering of the pH. Each of the three types of transitions described above occurs within a distinct pH range. From alkaline pH values down to about pH 4.5, only the type-1 transition is observed, and from pH 4.2 to pH 2.9, a pure type-2 transition is observed upon heating. At very low pH values, the spectral shift upon heating resembles the type-3 transition. As shown above, the type-1 and type-2 thermochromism of BR is correlated to equilibriums between the 560 nm state and either the 460 or 630 nm state. The nature of these spectral states is discussed later. Type-3 thermochromism is observed at very low pH values and resembles temperature-induced decay of the 630 nm state (blue membrane). The magnitude of spectral

6948 J. Phys. Chem. B, Vol. 112, No. 23, 2008

Neebe et al.

Figure 4. Temperature dependence of the absorption ratio 460 nm/ 560 nm for a BR-WT/PAA gel at pH 6.9.

Figure 3. Reflectance spectra of thermochromic light-adapted BRWT gelatine films. (top) Reflectance spectra of neutral BR-WT film (pH 6.9) at 20 °C (solid line) and 50 °C (dashed line). (middle) Reflectance spectra of acidic BR-WT film (pH 4.2) at 20 and 50 °C. (bottom) The color shifts for both transitions are given in the CIELAB system. For the neutral BR film a type-1 transition, and for the acidic film a type-2 transition is observed. In the type-1 transition, the color shifts to more red and more yellow components upon heating, whereas, in the type-2 transition, the color loses the red component only.

changes according to type-3 thermochromism is significantly lower than that for type-1 and type-2 thermochromism. Additionally, samples show rapid denaturation at temperatures >45 °C at pH values below 2.2. 3.2. Colorimetric Analysis of the Thermochromism of BR. In many potential technical applications of PM, the reflection spectra are more important than the transmission spectra. For this reason, we analyzed the temperature-dependent color changes of BR-WT quantitatively using the CIELAB color system.14 This colorimetric system provides an absolute color space, in which the color coordinates are arranged in such a way that the distance between two colors anywhere in the space corresponds to their physiological distance. It is a three-dimensional system with the coordinates a* (green/red balance), b* (blue/yellow balance), and L* (lightness axis). The color difference (∆ELab) is calculated as the geometrical distance between the threedimensional positions in the CIELAB color space. Figure 3 shows the temperature-dependent reflectance spectra of BR-WT gelatine films. For a neutral gelatine film of BRWT (pH 6.9) at 21 °C, we calculated the L*/a*/b* color coordinates to be 37/31/-19. Upon heating up to 50 °C, the color shifted to 39/33/-9. Therefore, the thermochromic effect

for a neutral PM preparation is composed of a slight increase in lightness, an increase in the red color component, given by a positive a* value, and a significant decrease in the blue color component, given by a negative b* value. The latter is causing the main effect in this thermochromic color change. In comparison to the neutral film, an acidic BR-WT film (pH 4.2) starts with a darker, more blue (lower b*) and more red (higher a*) color at 18 °C. The CIELAB coordinates are 26/34/-35. After heating to 50 °C, a color of 26/26/-32 is calculated. The main effect here is a loss in red color (decreasing positive a*). As the samples do not show much change in lightness during thermochromic switching, the color and the corresponding color changes can be plotted in the a*/b* plane of the color space (Figure 3, bottom). A color difference of 4 in CIELAB units is sufficient to be visually detectable. As the pH-dependent thermochromism of BR involves color differences of 10.3 and 8.0, we conclude that the color changes are suitable for visual detection in BR based materials. 3.3. Investigating the Origin of BR Thermochromism. Hypsochromic Shift. The temperature-dependent shift in the BR absorbance spectra from a species absorbing at 560 nm to a species absorbing at 460 nm has been observed in earlier studies. This transition has been reported to be reversible and pH-dependent, but its origin remained unclear.15,16 Furthermore, a similar spectral transition has been observed by direct titration of BR to higher bulk pH.15,17–19 Deprotonation of the Schiff base leads to a spectral transition from 560 to 460 nm absorbance with an observed pKa of 13.3 ( 0.3. The pKa of this transition is usually very high but is severely affected by structural changes in the environment of the chromophore. For example, it is reported that the pKa for Schiffbase deprotonation in wild-type BR is lowered by 3-4 pH units in each of the mutants D85A, D85N, D85C, and D85H.20 This shows that alterations of the counterion properties may strongly affect the pKa of the Schiff base. Therefore, temperature-induced structural changes in the counterion location are likely to influence the Schiff-base pKa in a similar manner and may thus provide a molecular basis for the pH-dependent thermochromism of BR. A temperature-induced hypsochromic spectral shift, related to a decrease in 560 nm absorption, is reported to be caused by the temperature dependence of the chromophore’s all-trans to 13-cis isomeric ratio in BR, but for temperatures up to 40 °C, the ratio remains unaffected.21 Therefore, we can exclude the equilibriums between the isomers B (all-trans retinal) and D (13-cis retinal) to be involved in thermochromism up to the mid temperature range. In Figure 4, the temperature dependence of the 460 nm state is plotted, and the temperature range for the type-1 transition

Thermochromism of Bacteriorhodopsin

J. Phys. Chem. B, Vol. 112, No. 23, 2008 6949

Figure 5. Temperature-dependent spectra of a BR-WT film on mica. A 655 nm species arises upon heating from 10 to 40 °C (inset). Obviously, this bathochromic population has nothing in common with the regular blue membrane, as the latter one accumulates a species absorbing at 630 nm.

can be identified. Starting with a neutral sample of dark-adapted BR-WT at 10 °C, we find a strong increase in 460 nm absorbance upon heating, with maximum increase near 60 °C. Since the 13-cis content (BR spectral D state, 548 nm maximum absorbance) begins to decrease distinctly and rapidly near 70 °C,21 we conclude that temperature-induced cis-trans isomerization is not involved in the thermochromic type-1 transition because the major spectral changes take place at temperatures below 60°. Bathochromic Shift. The appearance of bathochromically shifted absorbance states is related to the generation of the socalled “blue membrane”. Several types of blue membrane have been observed. Removal of cations22–25 as well as acid-mediated protonation of Asp85,26–28 part of the Schiff-base-complex (SBC) counterion, lead to the formation of blue membrane. Additionally, a temperature-induced bathochromic shift of BR absorbance spectra has been observed. Dark-adapted BR-WT is reported to reversibly populate a 630 nm absorbance state upon heating to temperatures above 70 °C when prepared in deionized water (pH 5.5-6).29 It was postulated that this effect is due to the release of divalent cations of BR at elevated temperatures, but cation release is not the only reason for bathochromic shifts of BR absorbance upon heating, as the formation of a 650 nm absorbance state was found upon heating of dark-adapted BR at pH 6 in high salt buffer.30 Furthermore, in thin film preparations of BR, a bathochromic spectral shift was found to occur upon heating to 140 °C, and was suggested to be caused by an equilibrium between the B and O state in BR photocycle.30 Indeed, we observed the rise of a bathochromic population, absorbing at wavelengths >655 nm, even in the low temperature range when BR-WT films, deposited onto mica, were heated to 40 °C (Figure 5). In the case of BR thermochromism, we conducted titration experiments with modified BR samples at 10 and 45 °C which give evidence that the temperature-induced bathochromic shift of BR absorbance is based on temperature-dependent changes in the protonation state of the Schiff base. In a first step, BR mutant E194Q was used, in which glutamic acid 194 is replaced by glutamine. In WT-BR, E194 is a titratable residue with 3.0 kcal/mol interaction energy to Asp85.31 Therefore, substituting E194 for alkaline glutamine affects the pKa of Asp85, the interacting residue of the Schiff base. Indeed, we find that the pH dependence of 630 nm absorbance is shifted toward smaller pH values compared to WT-BR (Figure 6, middle). In a second step, we modified the PM surface by acetylation of BR lysine residues, which causes a loss in positive charge at the PM surface and is reported to shift the pH dependence of

Figure 6. Titration curves. pH dependence of 630 nm absorbance at 10 °C (square symbols) and 45 °C (triangular symbols) of PAA gels of BR-WT (top), mutant E194Q (middle), and Ac-BR (bottom). Insets: Difference spectra (45-10 °C).

the protonation state of the protonated counterion without direct modification of the complex counterion.13 In our titration experiments with Ac-BR, we find that the bulk pH dependence of the 630 nm absorbance is shifted toward higher pH values compared to WT-BR, just as expected (Figure 6, bottom). As shown in Figure 6, both types of modification, E194Q and Ac-BR, shift the BR acid titration curves relative to BRWT, but just one modification affects BR thermochromism. The small insets in Figure 6 show the corresponding difference spectra, 45-10 °C, of pH-dependent 630 nm absorbance. For BR-WT, the temperature-induced absorbance change (45-10 °C) is found to reach a maximum at pH 2.9. Therefore, the preferred pH for the type-2 transition of BR-WT is pH 2.9. In the case of Ac-BR, changing the initial concentration of blue membrane leads to a similar course in the temperature-dependent difference spectrum (Figure 6, bottom, inset). We follow that the thermochromism of BR is independent of the initial concentration of the bathochromic species. On the other hand, a mutational change in the direct environment of the SBC counterion does affect thermochromism. For mutant E194Q, the temperature-dependent

6950 J. Phys. Chem. B, Vol. 112, No. 23, 2008

Neebe et al. analogies give strong evidence for deterioration of protein tertiary structure being the origin of BR thermochromism. 4. Conclusions

Figure 7. Solvent effect on BR-WT. Shown here is the difference spectrum obtained from a BR suspension (pH 2.9, 75 mM KCl, 25 °C), containing 10% ethanol, versus a reference BR suspension without ethanol. A rise of 460 nm as well as 630 nm absorbing states is observed. The effects of solvent exposition are similar to the thermochromic shifts observed upon heating.

difference spectrum is shifted toward lower pH, compared to BR-WT (Figure 6, middle, inset). Comparing the two effects, we conclude that the temperaturedependent absorbance changes in BR are controlled by the pKa of the SBC counterion and not by the initial concentration of the blue fraction. We generalize these results to the hypothesis that type-1, type-2, and type-3 thermochromism of BR are caused by temperature-dependent changes in the pKa of the SBC counterion. 3.4. Deterioration of BR Tertiary Structure and Thermochromism. The visual absorbance spectrum of BR is strongly dependent on chromophore-protein interactions as well as SBC counterion interactions, both causing the opsin shift.32 Changing these interactions will shift the chromophore’s absorbance in the visible. As shown above, BR thermochromism is based mainly on temperature-dependent changes in the pKa of the SBC counterion. Such pKa changes are related to structural changes in the protein. Heating PM through its premelting temperature is accompanied by several changes in BR secondary structure and trimer assembly which result in weakening of intramolecular forces, responsible for BR tertiary structure.33–40 Weakening of the tertiary structure is likely to affect the strong electrostatic coupling of the SBC and the counterion and provides a molecular basis for BR thermochromism. To get further insight into the correlation between conditions which lead to destabilization of BR tertiary structure and resulting changes in absorbance, we compared the thermally caused spectral changes to chemically induced absorbance shifts. The addition of low amounts of organic solvents to aqueous suspensions of PM is reported to affect the tertiary structure of BR in a reversible manner, causing spectral changes.15,41 In subsequent experiments, we observed deep analogies between spectral changes due to solvent exposition and the thermally caused changes. When BR-WT is suspended in an acidic medium (pH 2.9, 75 mM KCl), the spectral changes upon heating follow the type-2 thermochromism (rise of the bathochromic state). Keeping the sample at 25 °C, addition of ethanol (to a mole fraction of 10%), instead of heating, reversibly generates a 460 nm state as well as a bathochromic population, absorbing at 633 nm (Figure 7). These spectral changes resemble a superposition of features according to type-1 and type-2 transitions. Indeed, we observe that under several nondenaturating solvent-exposition conditions BR does reversibly populate a 460 nm state and a 630 nm state, similar to the thermally generated states. However, the quantitative distribution of states depends on the bulk pH in a different manner (not shown here). These

We find that membrane-integrated BR shows reversible thermochromism at physiological temperatures which is suitable for visual detection in optical applications. These temperaturedependent spectral absorbance changes are observed from 10 °C up to the denaturation temperature of the protein. Three different types of reversible spectral transitions are observed upon heating. The type-1 transition is characterized by a decrease in the 560 nm absorbance and a rise in the 460 nm absorbance, the type-2 transition, by a decrease in the 560 nm absorbance and a rise in the 630 nm absorbance, and the type-3 transition, by a decrease in the 630 nm absorbance and a rise in the 519 nm absorbance. In all types of spectral transition, we find isosbestic points, indicating two-state equilibriums. The spectral transitions depend on bulk pH. At neutral pH, a pure type-1 transition is observed. Below pH 4.5, the type-1 transition is no longer observed. From bulk pH 4.5 to pH 2.9, BR thermochromism is a pure type-2 transition. The type-2 transition is maximal at pH 2.9. Below pH 2.4, BR thermochromism turns into a pure type-3 transition. We suggest that reversible temperature-induced changes in the tertiary structure of the protein are the origin for the spectral shifts observed here. It is reasonable that structural changes influence the pKa of the SBC counterion which results in a shift either in the protonation state of Asp85 or the Schiff base. This suggestion is supported by the observation of similar spectral shifts upon nonthermal structural weakening of the protein by organic solvents. References and Notes (1) Oesterhelt, D.; Stoeckenius, W. Nat. New Biol. 1971, 233, 149. (2) Haupts, U.; Tittor, J.; Oesterhelt, D. Annu. ReV. Biophys. Biomol. Struct. 1999, 28, 367. (3) Grigorieff, N.; Ceska, T. A.; Downing, K. H.; Baldwin, J. M; Henderson, R. J. Mol. Biol. 1996, 259, 393. (4) Kimura, Y.; Vassylyev, D. G.; Miyazawa, A.; Kidera, A.; Matsushima, M.; Mitsuoka, K.; Murata, K.; Hirai, T.; Fujiyoshi, Y. Nature 1997, 389, 206. (5) Essen, L.-O.; Siegert, R.; Lehmann, W. D.; Oesterhelt, D. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11673. (6) Luecke, H.; Schobert, B.; Richter, H.-T.; Cartailler, J.-P.; Lanyi, J. J. Mol. Biol. 1999, 291, 899. (7) Sass, H. J.; Bu¨ldt, G.; Gessenich, R.; Hehn, D.; Neff, D.; Schlesinger, R.; Berendzen, J.; Ormos, P. Nature 2000, 406, 649. (8) Subramaniam, S.; Henderson, R. Nature 2000, 406, 653. (9) Oesterhelt, D.; Bra¨uchle, C.; Hampp, N. Q. ReV. Biophys. 1991, 24, 425. (10) Birge, R. R. Annu. ReV. Phys. Chem. 1990, 41, 683. (11) Hampp, N. Chem. ReV. 2000, 100, 1755. (12) Oesterhelt, D.; Stockenius, W. Methods Enzymol. 1974, 31, 667. (13) Maeda, A.; Takeuchi, Y.; Yoshizawa, T. Biochemistry 1982, 21, 4479. (14) Amerian Society for Testing and Materials, ASTM designation E308-01, 2001. (15) Oesterhelt, D.; Meentzen, M.; Schuhmann, L. Eur. J. Biochem. 1973, 40, 453. (16) Brouliette, C. G.; Muccio, D. D.; Finney, T. K. Biochemistry 1987, 26, 7431. (17) Druckmann, S.; Ottolenghi, M.; Pande, A.; Callender, R. H. Biochemistry 1982, 21, 4953. (18) Maeda, A.; Ogura, T.; Kitagawa, T. Biochemistry 1986, 25, 2798. (19) Balashov, S. P.; Govindjee, R.; Ebrey, T. G. Biophys. J. 1991, 60, 475. (20) Subramaniam, S.; Greenhalgh, D. A.; Khorana, H. G. J. Biol. Chem. 1992, 267, 25730. (21) Scherrer, P.; Mathew, M. K.; Sperling, W.; Stoeckenius, W. Biochemistry 1989, 28, 829. (22) Chang, C.-H.; Chen, J. G.; Govindjee, R.; Ebrey, T. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 396. (23) Liu, S. Y.; Ebrey, T. G. Photochem. Photobiol. 1987, 46, 557.

Thermochromism of Bacteriorhodopsin (24) Zhang, Y. N.; El-Sayed, M. A.; Bonet, M. L.; Lanyi, J. K.; Chang, M.; Ni, B.; Needleman, R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1445. (25) Birge, R. R.; Govender, D. S. K.; Izgi, K. C.; Tan, E. H. L. J. Phys. Chem. 1996, 100, 9990. (26) Mowery, P. C.; Lozier, R. H.; Chae, Q.; Tseng, I. W.; Taylor, M.; Stoeckenius, W. Biochemistry 1979, 18, 4100. (27) Fischer, U. C.; Towner, P.; Oesterhelt, D. Photochem. Photobiol. 1981, 33, 529. (28) Kimura, Y.; Ikegami, A.; Stoeckenius, W. Photochem. Photobiol. 1984, 40, 641. (29) Chang, C.-H.; Jonas, R.; Melchiore, S.; Govindjee, R.; Ebrey, T. G. Biophys. J. 1986, 49, 731. (30) Shen, Y.; Safinya, C. R.; Liang, K. S.; Ruppert, A. F.; Rothschild, K. J. Nature 1993, 366, 48. (31) Calimet, N.; Ullmann, M. J. Mol. Biol. 2004, 339, 571. (32) Houjou, H.; Inoue, Y.; Sakurai, M. J. Am. Chem. Soc. 1998, 120, 4459.

J. Phys. Chem. B, Vol. 112, No. 23, 2008 6951 (33) Jackson, M. B.; Sturtevant, J. M. Biochemistry 1978, 17, 911. (34) Brouillette, C. G.; Muccio, D. D.; Finney, T. K. Biochemistry 1987, 26, 7431. (35) Cladera, J.; Galisteo, M. L.; Dunach, M.; Mateo, P. L.; Padros, E. Biochim. Biophys. Acta 1988, 943, 148. (36) Arrondo, J. R.; Castresana, J. M.; Valpuesta, J. M.; Goni, F. M. Biochemistry 1994, 33, 11650. (37) Taneva, S. G.; Caaveiro, J. M. M.; Muga, A.; Coni, F. M. FEBS Lett. 1995, 367, 297. (38) Heyes, C D.; El-Sayed, M. A. Biochemistry 2001, 40, 11819. (39) Janoviak, H.; Kessler, M.; Oesterhelt, D.; Gaub, H.; Mu¨ller, D. J. EMBO J. 2003, 22, 5220. (40) Sonoyama, M.; Mitaku, S. J. Phys. Chem. B 2004, 108, 19496. (41) Mitaku, S.; Ikuta, K.; Itoh, H.; Kataoka, R.; Naka, M.; Yamada, M.; Suwa, M. Biophys. Chem. 1988, 30, 69.

JP7111389