Dynamics of Dangling Bonds of Water Molecules in pharaonis

Letter pubs.acs.org/JPCL

Dynamics of Dangling Bonds of Water Molecules in pharaonis Halorhodopsin during Chloride Ion Transportation Yuji Furutani,*,†,‡,§ Kuniyo Fujiwara,†,‡ Tetsunari Kimura,†,‡,⊥ Takashi Kikukawa,¶ Makoto Demura,¶ and Hideki Kandori# †

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan ‡ Department of Structural Molecular Science, The Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan § PRESTO and ⊥CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan ¶ Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan # Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan S Supporting Information *

ABSTRACT: Ion transportation via the chloride ion pump protein pharaonis halorhodopsin (pHR) occurs through the sequential formation of several intermediates during a photocyclic reaction. Although the structural details of each intermediate state have been studied, the role of water molecules in the translocation of chloride ions inside of the protein at physiological temperatures remains unclear. To analyze the structural dynamics of water inside of the protein, we performed time-resolved Fourier transform infrared (FTIR) spectroscopy under H2O or H218O hydration and successfully assigned water O−H stretching bands. We found that a dangling water band at 3626 cm−1 in pHR disappears in the L1 and L2 states. On the other hand, relatively intense positive bands at 3605 and 3608 cm−1 emerged upon the formation of the X(N) and O states, respectively, suggesting that the chloride transportation is accompanied by dynamic rearrangement of the hydrogen-bonding network of the internal water molecules in pHR. SECTION: Biophysical Chemistry and Biomolecules

H

Thr126 and Ser130 and the positively charged guanidium group of Arg123 stabilize a chloride ion at the binding site.16,17 The chloride ion release and uptake have been considered to occur in the transition from the X(N) to O and from the O to pHR′, respectively.4,18,19 pHR′ is a spectrally silent intermediate and considered to have very similar structure to the original state pHR. During these processes, the anion in the initial binding site is likely to be translocated to the cytoplasmic side; Lys215 and Thr218 are considered to act as acceptors of the translocated anion.13,17 The cytoplasmic side of pHR provides a less polar environment, and only one water molecule near Thr218 was found in the X-ray structure (Figure 1).15 Therefore, the transient movement of water molecules into the cytoplasmic space is expected to facilitate anion transport. Previously reported low-temperature FTIR spectroscopy has shown that the rearrangement of water molecules near the PSB occurs in the early intermediate states, K and L1.9,10 However, water motion in the later intermediates, especially the X(N) and O states, has not been studied.

alorhodopsin (HR) is a light-driven chloride ion pump protein discovered in Haloarchea.1 HR is an α-helical seven-transmembrane protein that binds an all-trans-retinal via the protonated Schiff base (PSB) linkage with the lysine residue on the seventh helix. The absorption maximum (λmax) is at around 575 nm, similar to that of the well-known light-driven proton pump protein bacteriorhodopsin (BR).2 Light absorption triggers the all-trans to 13-cis isomerization of the retinal chromophore, and several intermediate states [i.e., K, L1, L2, X(N), O, and HR′] are formed during the photocyclic reaction.3−5 pharaonis halorhodopsin (pHR), from Natronomonas pharaonis, tolerates low salinity conditions and has been studied by various physicochemical methods such as flash photolysis,3−5 Fourier transform infrared (FTIR) spectroscopy,5−11 electron paramagnetic resonance spectroscopy,12 transient grating spectroscopy,13 and X-ray crystallography.14,15 Extensive information on the kinetics and structure of pHR has been accumulated; however, the dynamics of water molecules during the ion pumping process remains unknown. Under aqueous conditions, a chloride ion is hydrated by several water molecules, and dehydration in a hydrophobic environment is energetically unfavorable. HR has an initial anion binding site near the PSB. The hydroxyl groups of © 2012 American Chemical Society

Received: August 29, 2012 Accepted: September 25, 2012 Published: September 25, 2012 2964

dx.doi.org/10.1021/jz301287n | J. Phys. Chem. Lett. 2012, 3, 2964−2969

The Journal of Physical Chemistry Letters

Letter

Figure 2. Intermediate spectra in the 1850−950 cm−1 region. The spectra were calculated from the decay-associated spectra assuming a unidirectional model without back reactions. The spectra on the left and right were measured under higher and lower chloride concentrations, respectively. The decay time constants are (a) 130 μs, (b) 550 μs, and (c) 2.90 ms for the former and (d) 320 μs, (e) 1.46 ms, and (f) 2.09 ms for the latter. The last intermediate spectra in the 1800−1700 cm−1 region are magnified in the insets of (c) and (f). Thin lines in the insets were measured with the D2O hydration condition. Dotted lines in (b), (e), and (f) are reproduced from the spectra in (c).

Figure 1. X-ray crystal structure of pHR (PDB code: 3A7K). Water molecules and chloride ions are shown as sky blue and yellow spheres, respectively. The all-trans-retinal chromophore is shown as a green stick model. The amino acid residues participating in chloride ion transportation are also shown as stick models in CPK colors with their residue numbers and names.

Here, we applied time-resolved FTIR (TR-FTIR) spectroscopy to pHR at pH 7 and 288 K with 12.5 μs intervals in the entire mid-infrared region. The ratio between the fractions of the X(N) and O intermediate states reportedly depends strongly on the chloride ion concentration.3,5 The O intermediate effectively accumulates under low salinity; in contrast, the X(N) intermediate accumulates at high salt concentrations. Thus, we prepared pHR film samples from suspensions having two chloride ion concentrations (0.2 and 5 mM NaCl). The final concentration of NaCl in the film sample was estimated to be ∼150 times of that in the suspension (Supporting Information). We denoted the former and latter conditions as the lower and higher salt conditions, respectively. The time-resolved spectral data sets were analyzed by singular value decomposition (SVD) and global exponential fitting; three exponentials were found to be sufficient for the reconstruction of the experimental data in both conditions. The obtained time constants (τ1, τ2, and τ3) were 0.13, 0.55, and 2.90 ms in the higher salt condition and 0.32, 1.46, and 2.09 ms in the lower salt condition, respectively. The intermediate spectra were calculated from the decay-associated spectra, assuming that the photoreaction proceeds unidirectionally without back reactions (Scheme 1). In the intermediate spectra, the bands from the intermediate states (P1, P2, and P3) appear in the positive direction, whereas those from the original state (P0; pHR) appear in the negative

direction. In Figure 2, the intermediate spectra in the higher and lower salt conditions are shown in the left and right panels, respectively. Both spectra sets are very similar to those reported by Hackmann et al.7 On the basis of the earlier report, the first and second spectra (Figure 2a and b) are considered to be the intermediate spectra of L1 and L2, respectively. The third spectrum looks similar to the second one, but a substantial difference in the amide I region is confirmed by superimposing it onto the second one (dotted line in Figure 2b). The C−C stretching bands of the retinal chromophore at 1170, 1191, and 1209 cm−1 are very similar. Therefore, the third intermediate has a 13-cis form, similar to the L1 and L2 intermediates. However, the amide I bands at 1648, 1674, and 1692 cm−1 differ from those in the second spectrum, suggesting that the third intermediate exhibits a different conformation from the L2 intermediate. On the basis of a previous flash photolysis experiment,4,5 we refer to this intermediate as X(N). In the higher salt condition, the O intermediate does not accumulate well. Therefore, the reaction scheme is as Shown in Scheme 2. The third intermediate spectrum, in the 1770−1700 cm−1 region, is expanded in the inset, where protonated carboxylate CO stretching and lipid ester CO stretching modes are expected to appear. The former downshifts in the D2O condition, owing to the H/D exchange from COOH to

Scheme 1

Scheme 2

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the O intermediate; however, these lipids are affected differently by the formation of the X(N) intermediate, where the signal appears on the negative side. This suggests that the O intermediate influences the lipid environment differently from the X(N) intermediate. Water has a broad O−H stretching vibration in the 3650− 2800 cm−1 region, and the frequency depends on the hydrogenbonding structure of water molecules. In the hydrogen-bonded network of water, a water molecule with a free O−H group, called a dangling bond, exhibits a frequency higher than 3600 cm−1.21,22 In a previous FTIR spectroscopy study of BR, a water O−H stretching band at 3643 cm−1 was assigned to the water molecule, the other O−H band of which forms a hydrogen bond with Asp85. Another water band at 3625 cm−1 was assigned to a water molecule close to Arg82. These bands are considered to be a useful probe of water molecules inside of the protein because of their peculiarly higher frequency with a narrower bandwidth than ordinary water O−H stretching bands. The intermediate spectra of pHR in the 3720−3550 cm−1 region after the removal of the transiently heated bulk water bands are shown in Figure 3. The 3605 (+)/3626 (−) cm−1

COOD, whereas the latter does not change. The positive band at 1733 cm−1 and negative band at 1718 cm−1 exhibit downshifts in the D2O condition, whereas the negative band at 1744 cm−1 is predominantly preserved. The spectral shape is similar to the L2 minus pHR difference spectrum at 250 K recorded earlier by low-temperature FTIR spectroscopy.11 According to that report, the band pair at 1735 (+)/1741 (−) cm−1 was assigned to Asp156, and the negative band at 1721 cm−1 was assigned to Glu234.11 The former was interpreted as a perturbation of the hydrogen bond in Asp156, and the latter was identified as the deprotonation of Glu234 upon the formation of the L2 intermediate at 250 K. At 288 K, this structural change was prolonged in the X(N) intermediate. In addition, the appearance of the H/D unexchangeable negative band at 1744 cm−1 indicates that lipid head groups are also affected by the formation of the X(N) intermediate, implying that a large structural change extending to the lipid−protein interface occurs. In the lower salt condition, the first intermediate spectrum resembles the spectrum observed in the higher salt condition, whereas the second and third spectra are different. The first spectrum can be assigned to the L1 intermediate. The second spectrum in the lower salt condition (solid line) can be superimposed on the third spectrum in the higher salt condition (dotted line), as shown in Figure 2e, implying that a similar intermediate state, X(N), is formed with a time constant of 1.46 ms. The third spectrum in the lower salt condition shows the characteristic features of the O intermediate state as rationalized below. Therefore, the reaction scheme at a lower salt concentration is as shown in Scheme 3. Scheme 3

The positive band at 1512 cm−1 in Figure 2f was assigned to the ethylenic CC stretching vibration of the retinal chromophore, whose frequency is known to correlate well with the absorption maximum (λmax).20 The reported λmax value of the O intermediate is about 610 nm, which is red-shifted from the original state, pHR (λmax = 575 nm), whose ethylenic mode is at 1525 cm−1. The positive bands at 1165 and 972 cm−1 are also signatures of the O intermediate state, indicating thermal reisomerization into the distorted all-trans configuration.5,7 The amide I bands at 1692, 1674, and 1656 cm−1 are more intense than those of the X(N) intermediate, whereas the positive 1648 cm−1 band disappears, indicating that different conformational changes occur in the O intermediate state.7 According to the X-ray crystal structure of the blue form of pHR, which is considered a model of the O intermediate state, a large deformation occurs around Thr126 in helix C, which constitutes the primary anion binding site.14 It appears that this structural change is probably correlated with the amide I changes. The negative band at 1718 cm−1 in the inset in Figure 2f exhibits a downshift in the D2O condition, whereas the positive band at 1741 cm−1 does so only partially. The former can be assigned to Glu234, as already described in the experiment in the higher salt condition. The disappearance of the corresponding positive band suggests that Glu234 remains deprotonated until the O intermediate state is formed. The appearance of the H/D unexchangeable positive band at 1741 cm−1 indicates that lipids are also affected by the formation of

Figure 3. Intermediate spectra in the 3720−3550 cm−1 region. The spectra on the left and right were measured at higher and lower chloride concentrations, respectively. The corresponding intermediate states are (a) L1, (b) L2, and (c) X(N) for the former and (d) L1, (e) X(N), and (f) O for the latter. The red and blue spectra were recorded under H2O and H218O hydration, respectively. The distortion from bulk water is subtracted using a band produced by curve fitting with two Gaussian functions (positive band at 3617 cm−1 with a HWHM of 63 cm−1 and negative band at 3221 cm−1 with a HWHM of 172 cm−1).

bands are observed in the X(N) intermediate in both higher (Figure 3c) and lower (Figure 3e) salt conditions. A relatively intense positive band is observed at 3608 cm−1 in the O intermediate with a negative band at 3626 cm−1 (Figure 3f). These bands are in the frequency region of a dangling bond of a water molecule. To assign these bands, we measured TR-FTIR data under H218O hydration, where the 18O isotope is expected to induce a downshift of the water O−H stretching mode (∼10 cm−1). The bands at 3605 (+)/3626 (−) cm−1 exhibit a downshift to 3598 (+)/3621 (−) cm−1 in the H218O hydration 2966



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1.5 times larger than that of the latter because of the relatively intense peak at 3608 cm−1, as already shown in the difference spectrum of the O intermediate. The accumulation of the O intermediate in the lower salt condition is obvious from the time traces of the ethylenic CC stretching mode of the retinal chromophore, as shown in Figure 4b. The 1512 cm−1 band is a signature of the O intermediate, which rises only in the lower salt condition. The 1525 cm−1 traces show the recovery of the original state, pHR, after laser irradiation; they exhibit almost the same kinetics in the higher (τ3 = 2.90 ms) and lower (τ3 = 2.09 ms) salt conditions. The 1558 cm−1 traces mainly show the accumulation and decay processes of the L2 or X(N) intermediate states. In addition to evidence from the intermediate spectra in Figure 3, we conclude that the intensity of the water dangling mode increases when the X(N) and O intermediate states are formed. How are water molecules rearranged during the pHR photocycle? A dangling bond of a water molecule is free from a hydrogen-bonded network. Amino acid residues inside of the protein provide a hydrogen bond donor and acceptor for stabilizing water molecules. The limited numbers of acceptor groups and spaces for water molecules inevitably yield a water molecule without a hydrogen bond. Therefore, the intensity of the O−H stretching vibrations of dangling bonds correlate well with the number of water molecules without hydrogen bonds inside of the protein. In the X(N) intermediate spectrum, the spectral shape of the bands at 3605 (+)/3626 (−) cm−1 is symmetrical with respect to the zero line (Figure 3c and e), implying that the number of dangling bonds does not change when the X(N) intermediate is formed. The origin of the 3626 cm−1 band is considered to be a water molecule near the initial anion binding site. Therefore, the environmental change of the water molecule may occur in X(N). On the other hand, the positive band at 3608 cm−1 is considerably larger than the negative band at 3626 cm−1 in the O intermediate spectrum. The disappearance of the chloride ion from the initial binding site may force some water molecules to cut their hydrogen bonds, which increases the water dangling bonds in O. In this study, we observed the dynamics of dangling bonds of water during the pHR photocycle by using TR-FTIR spectroscopy. We found that water molecules with dangling bonds are rearranged when the X(N) and O intermediates are formed. Further analysis using site-directed mutagenesis will clarify the specific locations of the water molecules in the intermediate states.

condition (Figure 3c), whereas similar bands in the lower salt condition exhibit downshift to 3595 (+)/3616 (−) cm−1 (Figure 3e). In the same way, the bands at 3608 (+)/3626 (−) cm−1 are downshifted to 3600 (+)/3618 (−) cm−1 (Figure 3f). The small downshift in the higher salt condition may be due to the incomplete replacement of H2O to H218O strongly hydrated to Na+ or Cl− ions in the hydrated sample. In any case, these bands are successfully assigned to water O−H stretching modes. In the L1 and L2 intermediate states, these bands are less obvious than those in the later intermediates (Figure 3a, b, and d); however, the negative band at 3626 cm−1 probably appears with lower intensity in the difference spectra of these states. Previously reported low-temperature FTIR spectroscopy for the K and L1 intermediates also detected the corresponding bands at 2683 cm−1 in the O−D stretching frequency region, which is expected to be observed at around 3626 cm−1 as an O−H stretching band.9,10 This band exhibited a slight halidetype dependency on the frequency and was assigned to a water molecule at the initial anion binding site of pHR. Our TR-FTIR data newly revealed positive bands at 3605 and 3608 cm−1 from water dangling bonds in the X(N) and O intermediates, respectively. In Figure 4a, we plot the time courses of the peak-to-peak height at 3605 and 3626 cm−1 for the lower (magenta) and higher (green) salt conditions. The amplitude of the former is

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EXPERIMENTAL SECTION Sample Preparation. The pHR sample was prepared as in the previous experiment with minor modifications.23 Depending on the sample quality, the His-tag-purified pHR was further purified with a Mono Q anion-exchange column. The samples used for the FTIR study had an optical purity of ∼1.8 (estimated from the ratio of the absorbances at 280 and 575 nm). Subsequently, the sample was reconstituted into PC liposomes with a lipid-to-protein molar ratio of 50:1 by adsorbing detergent micelles (n-dodecyl-β-D-maltopyranoside) into Bio-Beads (Bio-Rad). The reconstituted pHR sample was washed three times by a 2 mM sodium phosphate buffer (pH 7) with 0.2 or 5 mM NaCl to regulate the salt condition. Time-Resolved Step-Scan FTIR Spectroscopy. A 40 μL aliquot of the sample was dried on a CaF2 window (ϕ = 25 mm, thickness = 2 mm) and sealed by another window with a spacer consisting of a silicone rubber ring and a Parafilm sheet after

Figure 4. Time courses of dangling water O−H stretching bands and retinal ethylenic modes. (a) Peak-to-peak absorbance at 3605 and 3226 cm−1 and (b) absorbance at 1512, 1525, and 1558 cm−1 in the time-resolved FTIR spectra are plotted with respect to the time since laser irradiation. The experimental data are plotted by thin lines (a) and markers (b), and the fitting curves are plotted by thick lines (a,b). The plots for the lower and higher salt conditions are magenta and green, respectively. 2967



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hydration by nearly 8 μL drops of H2O containing 20% (v/v) glycerol to maintain moderate hydration. The sample was mounted in a temperature-controlled transmission cell holder (TFC-M25-3, Harrick), and the temperature was kept at 288 K by circulating water in a cooling thermostat bath (Alpha RA8, LAUDA). The time-resolved infrared spectra of the pHR samples were recorded by a FTIR spectrometer (VERTEX 80, Bruker Optics) equipped with a linearized mercury cadmium telluride detector. The photoreaction was triggered by a 532 nm laser pulse (∼1.0 mJ) with a duration of 10 ns and a repetition rate of 10 Hz from second-harmonic generation of a Nd:YAG laser (LS-2134LS, LOTIS-TII). The measurement was controlled by a digital delay generator (DG645, Stanford Research Systems). The time and spectral resolution were 12.5 μs and 8 cm−1, respectively. The light-induced transient signals were co-added 10 times at 1333 sampling points with 2000 time slices (25 ms in total) in the time-resolved step-scan mode, and 10−15 time-resolved interferograms were recorded and averaged for a given sample. SVD Analysis and Global Exponential Fitting for TR-FTIR Data. SVD was performed on an averaged spectral series obtained from each sample. The U spectra having singular values considerably larger than the others and whose corresponding V spectra contained the signal were used to estimate the number of exponentials and their rate constants to fit the data. Thereafter, the experimental data were globally fitted to exponential curves by using the estimated time constants to reduce the noise contribution. In all data sets, SVD analysis and the reductions in the standard deviation of the residuals were saturated at a three-exponential function. Thus, further analysis was performed according to the sequential model P1 → P2 → P3 → P0, where P0 is the unphotolyzed original state and Pi is a decay-associated state that may contain a few physically defined intermediates. By using the fitting results for a three-exponential function, the time constants τi and absorption differences between Pi and P0, which are denoted as intermediate spectra in this paper, were calculated. The data analyses was performed in MATLAB with personal scripts written by Dr. Vı ́ctor A. Lórenz-Fonfrı ́a.24

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supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS) to Y.F. (22770159, 22018030, 21026016) and T.K. (23687022). This work was also supported in part by the Joint Studies Program (2009−2010) of the Institute for Molecular Science. We also thank Vı ́ctor A. Lórenz-Fonfrı ́a for the use of a data analysis program. The authors would like to thank Enago (www.enago.jp) for the English language review.

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(1) Schobert, B.; Lanyi, J. K. Halorhodopsin is a Light-Driven Chloride Pump. J. Biol. Chem. 1982, 257, 10306−10313. (2) Váró, G. Analogies between Halorhodopsin and Bacteriorhodopsin. Biochim. Biophys. Acta 2000, 1460, 220−229. (3) Chizhov, I.; Engelhard, M. Temperature and Halide Dependence of the Photocycle of Halorhodopsin from Natronobacterium pharaonis. Biophys. J. 2001, 81, 1600−1612. (4) Hasegawa, C.; Kikukawa, T.; Miyauchi, S.; Seki, A.; Sudo, Y.; Kubo, M.; Demura, M.; Kamo, N. Interaction of the Halobacterial Transducer to a Halorhodopsin Mutant Engineered so as to Bind the Transducer: Cl− Circulation within the Extracellular Channel. Photochem. Photobiol. 2007, 83, 293−302. (5) Varo, G.; Brown, L. S.; Sasaki, J.; Kandori, H.; Maeda, A.; Needleman, R.; Lanyi, J. K. Light-Driven Chloride Ion Transport by Halorhodopsin from Natronobacterium pharaonis. 1. The Photochemical Cycle. Biochemistry 1995, 34, 14490−14499. (6) Guijarro, J.; Engelhard, M.; Siebert, F. Anion Uptake in Halorhodopsin from Natromonas pharaonis Studied by FTIR Spectroscopy: Consequences for the Anion Transport Mechanism. Biochemistry 2006, 45, 11578−11588. (7) Hackmann, C.; Guijarro, J.; Chizhov, I.; Engelhard, M.; Rödig, C.; Siebert, F. Static and Time-Resolved Step-Scan Fourier Transform Infrared Investigations of the Photoreaction of Halorhodopsin from Natronobacterium pharaonis: Consequences for Models of the Anion Translocation Mechanism. Biophys. J. 2001, 81, 394−406. (8) Hutson, M. S.; Shilov, S. V.; Krebs, R.; Braiman, M. S. Halide Dependence of the Halorhodopsin Photocycle as Measured by TimeResolved Infrared Spectra. Biophys. J. 2001, 80, 1452−1465. (9) Shibata, M.; Muneda, N.; Ihara, K.; Sasaki, T.; Demura, M.; Kandori, H. Internal Water Molecules of Light-Driven Chloride Pump Proteins. Chem. Phys. Lett. 2004, 392, 330−333. (10) Shibata, M.; Muneda, N.; Sasaki, T.; Shimono, K.; Kamo, N.; Demura, M.; Kandori, H. Hydrogen-Bonding Alterations of the Protonated Schiff Base and Water Molecule in the Chloride Pump of Natronobacterium pharaonis. Biochemistry 2005, 44, 12279−12286. (11) Shibata, M.; Saito, Y.; Demura, M.; Kandori, H. Deprotonation of Glu234 during the Photocycle of Natronomonas pharaonis Halorhodopsin. Chem. Phys. Lett. 2006, 432, 545−547. (12) Mevorat-Kaplan, K.; Weiner, L.; Sheves, M. Spin Labeling of Natronomonas pharaonis Halorhodopsin: Probing the Cysteine Residues Environment. J. Phys. Chem. B 2006, 110, 8825−8831. (13) Inoue, K.; Kubo, M.; Demura, M.; Kamo, N.; Terazima, M. Reaction Dynamics of Halorhodopsin Studied by Time-Resolved Diffusion. Biophys. J. 2009, 96, 3724−3734. (14) Kanada, S.; Takeguchi, Y.; Murakami, M.; Ihara, K.; Kouyama, T. Crystal Structures of an O-like Blue Form and an Anion-Free Yellow Form of pharaonis Halorhodopsin. J. Mol. Biol. 2011, 413, 162−176. (15) Kouyama, T.; Kanada, S.; Takeguchi, Y.; Narusawa, A.; Murakami, M.; Ihara, K. Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas pharaonis. J. Mol. Biol. 2010, 396, 564−579. (16) Sato, M.; Kikukawa, T.; Araiso, T.; Okita, H.; Shimono, K.; Kamo, N.; Demura, M.; Nitta, K. Ser-130 of Natronobacterium pharaonis Halorhodopsin is Important for the Chloride Binding. Biophys. Chem. 2003, 104, 209−216.

ASSOCIATED CONTENT

S Supporting Information *

Supporting results consisting of light-induced difference spectra of pHR in the higher and lower salt conditions and intermediate spectra in the 3720−3550-cm−1 region before the subtraction of bulk water contribution. Effect of the use of 20% glycerol for hydration on the photoreaction of pHR and the concentration and distribution of the anion in the hydrated film are also shown. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by Precursory Research for Embryonic Science and Technology (PRESTO) and the Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST). It was also 2968



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(17) Sato, M.; Kubo, M.; Aizawa, T.; Kamo, N.; Kikukawa, T.; Nitta, K.; Demura, M. Role of Putative Anion-Binding Sites in Cytoplasmic and Extracellular Channels of Natronomonas pharaonis Halorhodopsin. Biochemistry 2005, 44, 4775−4784. (18) Ludmann, K.; Ibron, G.; Lanyi, J. K.; Váró, G. Charge Motions during the Photocycle of pharaonis Halorhodopsin. Biophys. J. 2000, 78, 959−966. (19) Varo, G.; Needleman, R.; Lanyi, J. K. Light-Driven Chloride Ion Transport by Halorhodopsin from Natronobacterium pharaonis. 2. Chloride Release and Uptake, Protein Conformation Change, and Thermodynamics. Biochemistry 1995, 34, 14500−14507. (20) Aton, B.; Doukas, A. G.; Callender, R. H.; Becher, B.; Ebrey, T. G. Resonance Raman Studies of Purple Membrane. Biochemistry 1977, 16, 2995−2999. (21) Garczarek, F.; Gerwert, K. Functional Waters in Intraprotein Proton Transfer Monitored by FTIR Difference Spectroscopy. Nature 2006, 439, 109−112. (22) Kandori, H. Role of Internal Water Molecules in Bacteriorhodopsin. Biochim. Biophys. Acta 2000, 1460, 177−191. (23) Sato, M.; Kanamori, T.; Kamo, N.; Demura, M.; Nitta, K. Stopped-Flow Analysis on Anion Binding to Blue-Form Halorhodopsin from Natronobacterium pharaonis: Comparison with the AnionUptake Process during the Photocycle. Biochemistry 2002, 41, 2452− 2458. (24) Lórenz-Fonfría, V. A.; Kandori, H. Spectroscopic and Kinetic Evidence on How Bacteriorhodopsin Accomplishes Vectorial Proton Transport under Functional Conditions. J. Am. Chem. Soc. 2009, 131, 5891−5901.

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Dynamics of Dangling Bonds of Water Molecules in pharaonis

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