Anomalous pH Effect of Blue Proteorhodopsin - The Journal of

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Anomalous pH Effect of Blue Proteorhodopsin Keisuke Yamada,† Akira Kawanabe,†,§ Susumu Yoshizawa,‡ Kentaro Inoue,‡ Kazuhiro Kogure,‡ and Hideki Kandori*,† †

Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa-shi, Chiba 277-8564, Japan

‡

S Supporting Information *

ABSTRACT: Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (B-PR; λmax ≈ 490 nm) and green-absorbing PR (G-PR; λmax ≈ 525 nm). In this report, we present conversion of B-PR into G-PR using anomalous pH effect. B-PR in LC1-200, marine γ-proteobacteria, absorbs 497 and 513 nm maximally at pH 7 and 4, respectively, whose pH titration was reversible (pKa = 4.8). When pH was lowered from 4, the λmax was further red-shifted (528 nm at pH 2). This is unusual because blue shift occurs by chloride binding in the case of bacteriorhodopsin. Surprisingly, when pH was increased from 2 to 7, the λmax of this B-PR was further redshifted to 540 nm, indicating that green-absorbing PR (PR540) is created only by changing pH. The present study reports the conformational flexibility of microbial rhodopsins, leading to the switch of absorbing color by a simple pH change. SECTION: Biophysical Chemistry

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should be noted that the D97N mutant B-PR from Hawaiian Ocean Time Station variant HOT75m4 shows a similar pH titration curve.10 This suggests that the counterion is not simply composed of Asp97 but is more complex. In any case, a lowpH-associated spectral red shift can be interpreted in terms of protonation of the negatively charged counterion complex. Consequently, weakened conterion causes delocalization of a positive charge along the polyene chain. The pH titration was reversible between 4 and 10 (Figure 1b). Lowering of pH from 4 causes a further red shift of λmax (Figure 1c). This pH effect is unusual because a blue shift is normally observed at acidic pH due to the binding of a chloride ion, as previously reported for bacteriorhodopsin (BR)11 and sensory rhodopsin II (SRII).12,13 This fact suggests the complex hydrogen-bonding network in B-PR and is possibly related to the observed pKa value for D97N B-PR.10 Then, when we returned pH from pH 2 to 7, surprisingly, we observed a further red shift to 540 nm (Figure 1d). The pH titration is summarized in Figure 1e. The sample originally looks orange because of the λmax at 497 nm (Figure 2). pH titration was reversible between 4 and 10, the λmax being located between 513 and 495 nm, respectively. An anomalous pH effect was observed between 2 and 4 because the titration was never reversible (Figure 1e). The decrease in pH from 4 to 2 caused the 15 nm red shift, and the following pH increase from 2 to 7 caused the additional 12 nm red shift.

isual and microbial rhodopsins contain 11-cis or all-trans retinal inside the core of seven transmembrane helices, respectively.1 The retinal chromophore is bound to a lysine residue of the seventh helix via a protonated Schiff base linkage. The color-tuning mechanism is one of the important aspects in the rhodopsin field because the color of a common molecule, either 11-cis or all-trans retinal Schiff base, is determined by the surrounding amino acids of protein.2−4 The interaction between the chromophore and protein may be experimentally proved by site-directed mutagenesis, and many mutations were introduced for visual and microbial rhodopsins for better understanding of their color tunings. Color can be also changed by changing the pH if the titratable group is located near the retinal chromophore. Figure 1a shows a typical example for a blue-absorbing proteorhodopsin (B-PR),5−8 which was found for LC1-200, marine γproteobacteria Photobacterium sp. found from Sagami Bay of Japan (100 m depth). Here B-PR of LC1-200 was expressed in E. coli, and its absorption properties were examined for the detergent-solubilized sample. B-PR maximally absorbs the 500 nm light at neutral and alkaline pH, where the λmax at pH 10 is located at 495 nm. As pH is lowered, absorption spectra exhibit a red shift with an isosbestic point, and the λmax at pH 4 is located at 513 nm (Figure 1a). Such pH dependence is typical for microbial rhodopsins, and the mechanism of this spectral shift is well-explained by an equilibrium of protonation and deprotonation of the counterion of the retinal chromophore.9 The corresponding amino acid is Asp97 in B-PR of LC1-200, and the pKa of the titration was found to be 4.8 (Figure 1b). It © 2012 American Chemical Society

Received: January 7, 2012 Accepted: March 1, 2012 Published: March 1, 2012 800

dx.doi.org/10.1021/jz3000257 | J. Phys. Chem. Lett. 2012, 3, 800−804

The Journal of Physical Chemistry Letters

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Figure 1. Absorption properties of B-PR in LC1-200. (a) Visible absorption spectra of B-PR in LC1-200 at pH 4, 5, 6, and 10. The absorption spectra exhibit a single isosbestic point (502 nm), indicating that the two forms are in equilibrium. The λmax is located at 513 and 495 nm at pH 4 and 10, respectively. (b) Titration curve of B-PR at pH 4−10, where the λmax of B-PR is plotted versus pH unit. Open and filled circles show the measurements of increasing (from pH 4 to 10) and decreasing (from pH 10 to 4) pH, respectively. Identical λmax for both increasing and decreasing pH confirm the reversible titration, and a solid line represents the fitting curve according to the Henderson−Hasselbalch equation (pKa = 4.8). (c) From pH 4, pH is further decreased to 2 by the addition of HCl. B-PR exhibits spectral red shift, whose λmax is located at 528 nm. (d) From pH 2, pH is again increased to 7 by the addition of NaOH, where pH titration is never reversible but showing further red shift to 540 nm. (e) Summary of pH titration in the present study. The λmax values of PR in LC1-200 are plotted at various pH. Gray line reproduces the pH titration curve in panel b, where pH titration is reversible. In contrast, pH titration is irreversible at pH 5, the future experiments will clarify whether such pH effect is common for B-PR. Functional conversion of proteins in one family is important because it significantly helps understanding of the molecular mechanism. The mutations of Q105L B-PR and L105Q G-PR are the typical case, where a single amino acid replacement switched the absorbing colors.8 The first example of the functional conversion in microbial rhodopsins was the conversion of BR, a proton pump, into a chloride pump by a single amino acid D85T replacement.16 BR was also engineered to function as a negative phototaxis sensor like SRII by introducing three amino acids.17 These are the examples of functional conversions by mutations. Although halorhodopsin (HR), a chloride pump, has never been converted to a proton pump by mutations, HR pumps protons by the addition of azide.18 It is known that light-sensor proteins such as SRI and SRII pump protons in the absence of the transducer proteins.19,20 These are the examples of functional conversion by modifying interaction partners. The present study provides another example of functional conversion in microbial rhodopsins, where an anomalous pH effect converts B-PR into the green-type.

Figure 2. (a) Pictures of B-PR at pH 7. Left and right pictures correspond to the original B-PR (λmax = 497 nm) and PR540 (λmax = 540 nm), respectively. (b) Visible absorption spectra at the three colored points in Figure 1e, the original B-PR at pH 7 (497 nm), B-PR at pH 2 (λmax = 528 nm), and PR540 at pH 7 (λmax = 540 nm).

and 13-cis, 15-syn states,1 which could affect colors. According to the HPLC analysis, the chromophore configurations of B-PR and PR540 were predominantly all-trans (96% all-trans for B-PR and 84% all-trans for PR540; Figure 4b), indicating that the alltrans form is responsible for color tuning of blue and green absorbing forms. An interesting observation emerges that PR540 contains 9% 11-cis retinal. Although microbial rhodopsins containing the 11-cis chromophore had never been reported, we recently found that middle rhodopsin (MR) from Haloquadratum walsbyi contains 7.6 and 30.1% 11-cis form for dark-adapted and light-adapted conditions, respectively.14 The amount is not enough to contribute to color tuning, but the existence of the 11-cis form suggests more flexible chromophore binding pocket for PR540. The conversion of B-PR into green-absorbing PR only by changing pH (Figures 1 and 2) accompanies an irreversible step at aroud pH 2. This suggests that protonatable groups in this pH range are responsible for the unique protein conformational changes, leading to the irreversible color change. It is well known that the position 105 determines the absorbing color of PR because B-PR or G-PR possesses Gln or Leu, respectively, and Q105L B-PR and L105Q G-PR absorbs green and blue lights, respectively.8 Therefore, the local structure around the position 105 may be similar between Q105L B-PR and PR540, the latter of which is formed by pH-induced protein

Figure 3. (a) Relaxation of PR540 to the original B-PR, where absorbance at 540 nm is monitored with time at pH 7 and 20 °C. Solid line represents a single exponential fitting curve, whose time constant (τ1/e) is 8.3 × 103 min (5 days and 19 h). (b) Visible absorption spectra of B-PR at pH 7. Dashed and solid lines represent the spectra of the original B-PR (λmax = 497 nm) and PR540 (λmax = 540 nm), respectively. Red line corresponds to the spectrum of PR540 after 6 months at 20 °C, indicating that PR540 is completely returned to the original B-PR. Note that the red line was measured after centrifuge (21 500g, 1 min) because some aggregates were formed during 6 months, which does not happen for the original B-PR. Consequently, the red line is multiplied by 1.3 for comparison, where 24% protein is lost probably because of less stability of PR540. (See Figure 4a.) 802



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Figure 4. (a) Thermal stability of B-PR (black line) and PR540 (red line). The samples are kept at 85 °C (pH 7.0), and the bleach by thermal decomposition is monitored versus incubation time. Note that the samples were centrifuged (21 500g, 1 min), because aggregates were formed by thermal decomposition. Three independent measurements are averaged. (b) HPLC analysis of the retinal chromophore extracted from B-PR (black line) and PR540 (red line). A high-performance liquid chromatograph was equipped with a silica column, and extraction of retinal oxime from the sample was carried out by hexane after denaturation by methanol and 500 mM hydroxylamine at 4 °C. After the extraction, retinal oxime exists in 15syn (s) and 15-anti (a) form, and the molar composition of retinal isomers was calculated from the areas of the peaks in the HPLC patterns. See Table 1 in the Supporting Information in more detail.

Absorbing lights of visual and microbial rhodopsins presumably depend on the chromophore−protein interaction of the 11-cis and all-trans retinal, respectively. The λmax of microbial rhodopsins distributes between 490 and 590 nm, although visual rhodopsins can absorb bluer lights.1 This difference partially originates from the chromophore configuration between the 11-cis and all-trans forms because the protonated Schiff base of the all-trans retinal absorbs 15−25 nm red-shifted light compared with that of the 11-cis retinal in the same clay interlayers.21 Linear π-conjugation of the all-trans form in microbial rhodopsins probably tends to absorb green to red light, which is in turn disadvantageous to absorb blue light, an environment of deep ocean. Presumably, there is a specific mechanism to maintain the λmax of B-PR at ∼490 nm. Biological relevance of the present finding is not clear because such a strong acid (pH 2) is never physiological. However, the possible switch of B-PR to the green type by a simple acidification suggests the presence of the specific protein structure to absorb blue light. The water-containing hydrogenbonding network plays a crucial role in the Schiff base region of microbial rhodopsins,1,22 and we infer that protonation of a group such as carboxylate drives conformation changes of the retinal binding pocket, leading to the transition to the greenabsorbing type. In this regard, structural analysis of PR540 is intriguing, and the FTIR study of PR540 is our future focus.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to H.K (20108014, 22247024) and Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists to A.K.

(1) Kandori, H. Retinal Binding Proteins. In Cis-Trans Isomerization in Biochemistry; Wiley-VCH: Weinheim, Germany, 2006; pp 53−75. (2) Kochendoerfer, G. G.; Lin, S. W.; Sakmar, T. P.; Mathies, R. A. How Color Visual Pigments Are Tuned. Trends Biochem. Sci. 1999, 24, 300−305. (3) Hoffmann, M.; Wanko, M.; Strodel, P.; Kö nig, P. H.; Frauenheim, T.; Schulten, K.; Thiel, W.; Tajkhorshid, E.; Elstner, M. Color Tuning in Rhodopsins: The Mechanism for the Spectral Shift between Bacteriorhodopsin and Sensory Rhodopsin II. J. Am. Chem. Soc. 2006, 128, 10808−10818. (4) Coto, P. B.; Strambi, A.; Ferre, N.; Olivucci, M. The Color of Rhodopsins at the ab initio Multiconfigurational Perturbation Theory Resolution. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17154−17159. (5) Béjà, O.; Aravind, L.; Koonin, E. V.; Suzuki, M. T.; Hadd, A.; Nguyen, L. P.; Jovanovich, S. B.; Gates, C. M.; Feldman, R. A.; Spudich, J. L.; et al. Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea. Science 2000, 289, 1902−1906. (6) Béjà, O.; Spudich, E. N.; Spudich, J. L.; Leclerc, M.; DeLong, E. F. Proteorhodopsin Phototrophy in the Ocean. Nature 2001, 411, 786−789. (7) Fuhrman, J. A.; Schwalbach, M. S.; Stingl, U. Proteorhodopsins: An Array of Physiological Roles? Nat. Rev. Microbiol. 2008, 6, 488− 494. (8) Man, D.; Wang, W.; Sabehi, G.; Aravind, L.; Post, A. F.; Massana, R.; Spudich, E. N.; Spudich, J. L.; Béjà, O. Diversification and Spectral Tuning in Marine Proteorhodopsins. EMBO J. 2003, 22, 1725−1731. (9) Yamada, K.; Kawanabe, A.; Kandori, H. Importance of Alanine at Position 178 in Proteorhodopsin for Absorption of Prevalent Ambient Light in the Marine Environment. Biochemistry 2010, 49, 2416−2423. (10) Wang, W. W.; Sineshchekov, O. A.; Spudich, E. N.; Spudich, J. L. Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin. J. Biol. Chem. 2003, 278, 33985−33991. (11) Dér, A.; Száraz, S.; Tóth-Boconádi, R.; Tokaji, Z.; Keszthelyi, L.; Stoeckenius, W. Alternative Translocation of Protons and Halide Ions by Bacteriorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4751− 4755. (12) Chizhov, I.; Schmies, G.; Seidel, R.; Sydor, J. R.; Lüttenberg, B.; Engelhard, M. The Photophobic Receptor from Natronobacterium

ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, pH titration after the formation of PR540, amino acid sequences of B-PR from Hawaiian Ocean Time Station variant HOT75m4, B-PR from LC1-200 and GPR, and HPLC analysis of the retinal configuration of B-PR and PR540 are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address §

Department of Physiology, Osaka University.

Notes

The authors declare no competing financial interest. 803



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pharaonis: Temperature and pH Dependencies of the Photocycle of Sensory Rhodopsin II. Biophys. J. 1998, 75, 999−1009. (13) Shimono, K.; Kitami, M.; Iwamoto, M.; Kamo, N. Involvement of Two Groups in Reversal of the Bathochromic Shift of pharaonis Phoborhodopsin by Chloride at Low pH. Biophys. Chem. 2000, 87, 225−230. (14) Sudo, Y.; Ihara, K.; Kobayashi, S.; Suzuki, D.; Irieda, H.; Kikukawa, T.; Kandori, H.; Homma, M. A Microbial Rhodopsin with a Unique Retinal Composition Shows Both Sensory Rhodopsin II and Bacteriorhodopsin-Like Properties. J. Biol. Chem. 2011, 286, 5967− 5976. (15) Hillebrecht, J. R.; Galan, J.; Rangarajan, R.; Ramos, L.; McCleary, K.; Ward, D. E.; Stuart, J. A.; Birge, R. R. Structure, Function, and Wavelength Selection in Blue-Absorbing Proteorhodopsin. Biochemistry 2006, 45, 1579−1590. (16) Sasaki, J.; Brown, L. S.; Chon, Y. S.; Kandori, H.; Maeda, A.; Needleman, R.; Lanyi, J. K. Conversion of Bacteriorhodopsin into a Chloride Ion Pump. Science 1995, 269, 73−75. (17) Sudo, Y.; Spudich, J. L. Three Strategically Placed HydrogenBonding Residues Convert a Proton Pump into a Sensory Receptor. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16129−16134. (18) Váró, G.; Brown, L. S.; Needleman, R.; Lanyi, J. K. Proton Transport by Halorhodopsin. Biochemistry 1996, 35, 6604−6611. (19) Bogomolni, R.; Stoeckenius, W.; Szundi, I.; Perozo, E.; Olson, K. D.; Spudich, J. L. Removal of Transducer HtrI Allows Electrogenic Proton Translocation by Sensory Rhodopsin I. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10188−10192. (20) Sudo, Y.; Iwamoto, M.; Shimono, K.; Sumi, M.; Kamo, N. Photo-Induced Proton Transport of Pharaonis Phoborhodopsin (Sensory Rhodopsin II) Is Ceased by Association with the Transducer. Biophys. J. 2001, 80, 916−922. (21) Furutani, Y.; Ido, K.; Sasaki, M.; Ogawa, M.; Kandori, H. Clay Mimics Color Tuning in Visual Pigments. Angew. Chem., Int. Ed. 2007, 46, 8010−8012. (22) Kandori, H. Role of Internal Water Molecules in Bacteriorhodopsin. Biochim. Biophys. Acta 2000, 1460, 177−191.

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Anomalous pH Effect of Blue Proteorhodopsin - The Journal of

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