THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 35, pp. 23533–23541, August 29, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Salinibacter Sensory Rhodopsin
SENSORY RHODOPSIN I-LIKE PROTEIN FROM A EUBACTERIUM *Received for publication, April 18, 2008, and in revised form, June 5, 2008 Published, JBC Papers in Press, June 19, 2008, DOI 10.1074/jbc.M802990200

Tomomi Kitajima-Ihara‡, Yuji Furutani§, Daisuke Suzuki‡, Kunio Ihara¶, Hideki Kandori§, Michio Homma‡,
and Yuki Sudo‡1
From the ‡Division of Biological Science, Graduate School of Science and the ¶Center for Gene Research, Nagoya University,
Chikusa-Ku, Nagoya, 464-8602, Japan and the §Department of Materials Science and Engineering, Nagoya Institute of Technology,
Showa-ku, Nagoya, 466-8555, Japan

Microorganisms are subjected to a variety of environmental
stimuli to which they respond and adapt. Sensory rhodopsin I(SRI),2 a photoactive membrane-embedded retinylidene pro-* This work was supported by Grants 19042013 and 19045015 (to Y. F.),
19370067 and 20050015 (to H. K.), and 19870010 (to Y. S.) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The
costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed. Tel.: 81-52-789-2993; Fax:
81-52-789-3001; E-mail: 4sudo@bunshi4.bio.nagoya-u.ac.jp.
2
The abbreviations used are: SRI, sensory rhodopsin I; SrSRI, S. ruber SRI;HsSRI, H. salinarum SRI; NpSRII, N. pharaonis SRII (also known as pharaonis
phoborhodopsin, ppR); HtrI, halobacterial transducer protein I; HsHtrI, H.
salinarum HtrI; SrHtrI, S. ruber HtrI; NpHtrII, N. pharaonis HtrII; BR, bacteriorhodopsin; SrSRIK, SrSRI K intermediate; SrSRIM, SrSRI M intermediate; HPLC,
high performance liquid chromatography; FTIR, Fourier transform infrared
spectroscopy; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid;
CAPS, 3-(cyclohexylamino)propanesulfonic acid.

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tein consisting of seven transmembrane helices, functions as a
receptor regulating both negative and positive phototaxis in thearchaeon Halobacterium salinarum (1, 2). The original state of
SRI and its long-lived photointermediate (the M-intermediate)
are important for positive and negative phototaxis, respectively,
and have absorption maxima at 587 and 373 nm, respectively.
Sensory rhodopsin II (SRII, also known as phoborhodopsin) is anegative phototaxis receptor in haloarchaea, including H. salinarum and Natronomonas pharaonis. Those are called HsSRII
and NpSRII, respectively, and their absorption maxima are atabout 500 nm (3). Thus, haloarchaea are attracted to light withwavelengths longer than 520 nm, and they avoid light withwavelengths shorter than 520 nm because of the functions of
SRI and SRII (2). Light ⬎520 nm can activate the ion pumping
rhodopsins, bacteriorhodopsin (BR), and halorhodopsin (HR),to obtain light energy, and cells avoid light of shorter wavelengths that contain harmful near-UV radiation (2).
HsSRI and NpSRII form 2:2 signaling complexes with theircognate halobacterial transducer proteins (Htr), HsHtrI and
NpHtrII, in membranes, and transmit light signals throughchanges in protein-protein interactions (4). The excitation lightabsorbed by HsSRI and NpSRII triggers trans-cis isomerizationof the retinal chromophore that is covalently bound to a conserved lysine residue via a protonated Schiff base linkage (5, 6).
This photoexcitation results in the sequential appearance ofvarious photointermediates (K, L, M, and O) followed by areturn to the unphotolyzed form of the protein (2, 7). The linear
cyclic photochemical reaction is referred to as the photocycle.
In the dark (original) state of NpSRII, it has been reported
that two specific hydrogen bonds between NpSRII and NpHtrII(8, 9), the deprotonation of Asp-193NpSRII (10) and the HAMP
domain of NpHtrII (11), are important for their interactions.
Upon formation of the K-intermediate, light activates the photoisomerization of retinal in NpSRII, which leads to steric constraint between Thr-204NpSRII and C14-H in the retinal chromophore (12, 13) and to alteration of the O-H stretchingvibration of Thr-204 (14, 15), as revealed by phototaxis and
FTIR experiments. Upon formation of the active M-intermediate, helix-F of NpSRII moves toward NpHtrII, as revealed byelectron paramagnetic resonance spectroscopy (16) and chemical modification (17), which leads to helix rotation and/or helix
movements of NpHtrII (18). At its distal end, activated NpHtrII
binds a cytoplasmic histidine kinase, CheA. CheA phosphorylates a phosphoregulator (CheY) that binds to the flagellar
motor switch and controls the swimming reversal probability of
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Halobacterium salinarum sensory rhodopsin I (HsSRI), a
dual receptor regulating both negative and positive phototaxis
in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterialtransducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a
receptor regulating negative phototaxis. Compared with HsSRI,
the signal relay mechanism of SRII is well characterized because
SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions andis much more resistant to detergents. Two genes encoding SRI
homologs were identified from the genome sequence of the
eubacterium Salinibacter ruber. Those sequences are distantly
related to HsSRI (⬃40% identity) and contain most of the aminoacid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), wecloned and expressed them in Escherichia coli. One of them
(SrSRI) was expressed well as a recombinant protein having alltrans retinal as a chromophore. UV-Vis, low-temperature UVVis, pH-titration, and flash photolysis experiments revealed thatthe photochemical properties of SrSRI are similar to those of
HsSRI. In addition to the expression system, the high stability of
SrSRI makes it possible to prepare large amounts of protein andenables studies of mutant proteins that will allow new
approaches to investigate the photosignaling process of
SRI-HtrI.

SRI from a Eubacterium

EXPERIMENTAL PROCEDURES
Plasmids and Strains—S. ruber (a kind gift from Dr. DyallSmith) was grown aerobically at 40 °C at pH 7.0 in medium withthe following composition (g/liter): NaCl, 195; MgSO4䡠7H2O,
25; MgCl2䡠6H2O, 16.3; CaCl2䡠2H2O, 1.25; KCl, 5.0; NaHCO3,
0.25; NaBr, 0.625; and yeast extract (Difco), 1.0. Cells were harvested by centrifugation and were stored at ⫺80 °C. Genomic
DNA was prepared using the method of Marmur (24). E. coli
DH5␣ was used as a host for DNA manipulation, andBL21(DE3) was used for expressing the genes. For DNA manipulation, the sensory rhodopsin I gene (SRU_2511) was amplified using PCR from the genomic DNA of S. ruber. The forward
primer 5⬘- CATATGGACCCGATTACGATCGTCTA-3⬘ andthe reverse primer 5⬘-CTCGAGGGCGGCAGACGTGACTGTCGC-3⬘ were designed (underlining indicates the restriction sites for NdeI and XhoI, respectively). The stop codon wasdeleted during amplification. The NdeI and XhoI fragment was
ligated to NdeI and XhoI sites of pET21c(⫹) vector (Novagen,Madison, WI). Consequently, the plasmid encodes six histidines at the C terminus, and it was named pTK001. This cloning strategy resulted in the following N- and C-terminal peptide
sequences: 1MDPI----TSAA239LEHHHHHH. The constructed
plasmid was analyzed using an automated sequencer to confirm
the expected nucleotide sequence. Another SRI gene,
SRU_2579, was amplified and cloned using almost the same
strategy as SRU_2511, except for the use of the restriction site
NheI instead of NdeI.
Protein Expression, Purification, and Reconstitution into PG
Liposomes—Cells were grown in LB medium supplemented
with ampicillin (final concentration of 100 ␮g/ml). E. coli
BL21(DE3) harboring pTK001 was grown to an A660 of 0.3– 0.5
in a 30 °C incubator, followed by the addition of 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside and 10 ␮M all-trans retinal.
Cells were harvested at 3-h postinduction by centrifugation at

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4 °C, resuspended in buffer (50 mM MES, pH 6.5) containing 4 M
NaCl, and disrupted by sonication. Cell debris was removed bya low speed centrifugation (5,000 ⫻ g, 10 min, 4 °C). For solubilization of the membranes, 1% (w/v) n-dodecyl-␤-D-maltoside (DM) was added, and the suspension was incubated overnight at 4 °C. The solubilized membranes were isolated by highspeed centrifugation (100,000 ⫻ g, 30 min, 4 °C), and the supernatant was incubated with Ni-nitrilotriacetic acid (NTA)
superflow (Qiagen, Valencia, CA) for 30 min. Thereafter, theNi-NTA resin was poured into a chromatography column andwas washed extensively with buffer (50 mM MES, pH 6.0) containing 4 M NaCl, 20 mM imidazole, and 0.1% w/v DM to remove
unspecifically bound proteins. The histidine-tagged proteins
were then eluted with buffer (0.1% DM, 4 M NaCl, 50 mM MES,
pH 6.0, 300 mM imidazole). The purified SrSRI sample was thenreconstituted into PG liposomes. For reconstitution of L-␣phosphatidylglycerol (Sigma) lipids, the lipids were dissolved inchloroform, and a thin lipid film on the wall of a flask was
prepared by careful evaporation of the solvent. SrSRI in buffer(0.1% DM, 1 M NaCl, 10 mM MES, pH 6.0, 100 mM imidazole)
was added (at an SrSRI:PG molar ratio of 1:50), and the suspension was gently stirred for 30 min at room temperature. Reconstitution by detergent removal was performed using Bio-Beads
SM-2 (Bio-Rad), as previously described (25).
The expression plasmid of HsSRI was constructed essentiallyas described previously (9). HsSRI, with a tag of six histidines at
the C-terminal end, was expressed in E. coli BL21(DE3). Preparation of crude membranes and purification of protein weredone as described previously (25).
Room Temperature and Low Temperature UV-Vis Spectroscopy and HPLC Analysis—Room temperature UV-Vis spectra
were obtained using a UV2400PC spectrophotometer with an
ISR240A integrating sphere (Shimadzu, Kyoto, Japan). The PGreconstituted sample was washed and resuspended in six buffers (citric acid, MES, HEPES, MOPS, CHES, and CAPS, whose
concentrations were 10 mM each) with 1 M NaCl. The buffer
composition had the same buffer capacity over a wide range ofpH values (2–9). pH values of the sample suspensions were
measured using an F-55 pH-meter with a 9669-C10 glass electrode (HORIBA, Kyoto, Japan).
Low-temperature UV-Vis spectra were measured using a
V-550DS (JASCO, Tokyo, Japan) spectrophotometer with acryostat (Optistat DN, Oxford, UK) and a temperature controller (ITC 502, Oxford, UK) with liquid nitrogen as the coolant, as
described previously (26). The PG-reconstituted sample with 5
mM NaCl and 2 mM phosphate buffer (pH 7.0) was placed on a
BaF2 window, and then was dried to a film under reduced pressure with an aspirator. The films were hydrated with water.
SrSRIK minus SrSRI difference spectra were measured at 77 K,
as follows: Illumination of the SrSRI film with ⬎450 nm light for
2 min, after which at pH 7.0 and 77 K converted SrSRI to
SrSRIK, and subsequent illumination with ⬎640 nm light for 1
min forced SrSRIK to revert to SrSRI. SrSRIM minus SrSRI difference spectra were measured at 200 K and pH 7.0 as follows:
To convert SrSRI to SrSRIM, the sample was irradiated with
⬎480 nm light for 2 min, after which a subsequent 1-min illumination with UV light-changed SrSRIM back to SrSRI. The
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the cell (19, 20). Thus, compared with HsSRI, the signal relay
mechanism of NpSRII is well characterized by various methods(21), because NpSRII is much more stable than H. salinarum
SRII (22), especially in dilute salt solutions, and is much moreresistant to detergents whereas HsSRI is unstable under thoseconditions. Therefore, little is known about the molecularmechanism(s) of interactions between SRI and HtrI, about
structural changes, or about the signal relay mechanism of positive phototaxis.
New genes encoding the sensory rhodopsin I-like protein
have been recently discovered through genome sequencing of
S. ruber (23). We present here the first characterization of SRI
from the eubacterium S. ruber (SrSRI). SrSRI has all-trans retinal as a chromophore, has a longer absorption maximum (558nm) than does SRII (500 nm), and has a slower photocycle thanthe light-driven ion-pumping rhodopsins (BR, HR, and proteorhodopsin), indicating similarities to the prototypic SRI(HsSRI) (1). SrSRI could be expressed in Escherichia coli andshows very high stability, even in detergent micelles, making it
possible to prepare large amounts of protein. This also allows
preparation of mutant SrSRI, which will allow new approaches
to investigate the photosignaling process in the SRI-HtrI
system.

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RESULTS AND DISCUSSION
Two Sensory Rhodopsin I-like Proteins in the Genome of
S. ruber—The genome sequence of S. ruber contains two genes
coding for sensory rhodopsin I (SRU_2511, which are named
SrSRI, and SRU_2579). To date, these are the only examples(other than sensory proteorhodopsin from ␥-proteobacteria) ofan SRI gene discovered outside the Archaea (28). However, theproperties of sensory proteorhodopsin have not been identified
yet, because it is not expressed in E. coli, Pichia pastoris, or
H. salinarum. The sequences of SRU_2511(SrSRI) and
SRU_2579 are distantly related to HsSRI (⬃40% identity) (29)and contain most of the amino acid residues (Asn-165, His-166,
Asp-201, Arg-215) identified as necessary for function (30 –33)
(Fig. 1), except for the equivalent of Tyr-163 in SRU_2511. Fig.
1 shows the secondary structure of SRI-like proteins from S. ruber. It is well known that the sensory rhodopsins have seventransmembrane domains and that retinal is the chromophore.
A lysine (Lys-205) and an aspartate residue (Asp-72), predicted
to be necessary for stabilization of the Schiff base and for thebinding between protein and retinal, are completely conserved
among SRIs (Fig. 1). Unexpectedly, His-166, one of the mostimportant residues for active site proton transfer and phototaxis signaling (31) in HsSRI is replaced by Tyr in one of thesensory rhodopsin I-like proteins from S. ruber (SRU_2511).
These genes, SRU_2511 and SRU_2579, which have a downstream sequence are immediately followed by the same second
gene in a probable operon that is under the control of the samepromoter as rhodopsin. These putative transducer genes(SRU_2510, Htr for SRU_2511, and SRU_2580, Htr for
SRU_2579) encode a ⬃650-residue protein with two transmembrane domains at the N-terminal portion followed by anextensive domain with primarily hydrophilic residues. This
topology is similar to that of the Htr transducers from thearchaeon H. salinarum (34). The eubacterium, S. ruber, also
has two light-driven ion pumps, Xanthorhodopsin and a Halorhodopsin-like protein, making an energy source for living cells(23, 35). The sensory rhodopsins would function as a positive
phototaxis sensor against longer wavelengths of light (where
ion pumping rhodopsins can utilize light energy) and as a negative phototaxis sensor against shorter wavelengths of light(which contains harmful UV).
Protein Expression, Purification, and Absorption Spectrum—
To determine whether the genes encode functional proteins,
we cloned and expressed the two sensory rhodopsins,
SRU_2511 and SRU_2579, in E. coli BL21(DE3) cells. Using
PCR, we obtained a fragment containing the full-length of the
SRU_2511 and SRU_2579 gene coding regions. An NdeI site
was created at the translation initiation site in the PCR productusing an oligonucleotide primer containing the NdeI site. A
XhoI site was created, and the stop codon was deleted during
the amplification. The NdeI and XhoI fragment of the PCR
product was cloned into the NdeI and XhoI sites of pET21c(⫹).
Induction of E. coli BL21(DE3) with 0.5 mM isopropyl-1-thio␤-D-galactopyranoside caused growth inhibition, suggesting
they expressed the desired protein. Actually, upon addition ofretinal, a color change of the SRU_2511-transformed cells wasrecognized, and the color was definitely distinct from cells
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difference spectrum was calculated from the spectra before andafter the illumination.
High performance liquid chromatography (HPLC) analysis
was performed as described previously (27). The PG-reconstituted sample was analyzed in buffer (1 M NaCl, 50 mM Tris-Cl,
pH 7.0). The HPLC system consisted of a PU-2080 pump and a
UV-2075 UV/Vis detector (JASCO). The chromatograph was
equipped with a silica column (6.0 ⫻ 150 mm; YMC-Pack SIL),
the solvent was 12% (v/v) ethyl acetate and 0.12% (v/v) ethanol
in hexane, and the flow rate was 1.0 ml/min. Extraction of retinal oxime from the sample was carried out by hexane after
denaturation by methanol and 500 mM hydroxylamine at 4 °C.
The molar composition of retinal isomers was calculated fromthe areas of the peaks monitored at 360 nm. Assignment of eachpeak was performed by comparing it with the HPLC patternfrom retinal oximes of authentic all-trans- and 13-cis-retinals.
For the light-adapted SrSRI, the sample was irradiated with
⬎480 nm light for 1 min. Two independent measurements
were averaged.
Time-resolved Laser Spectroscopy—The purified sample in
DM detergent micelles was resuspended in 50 mM Tris-HCl,
pH 7.0, 0.1% DM, and 1 M NaCl. Flash-induced absorptionchanges in the 300 –700-nm region in the millisecond to second
time window with a 10-ms interval were acquired using a commercial flash photolysis system (Hamamatsu Photonics K. K.,
Hamamatsu, Japan), which consisted of a CCD linear detector(Photonic Multichannel Spectral Analyzer PMA-11 C8808 –01), a CW xenon lamp L8004 as a light source and a sample
room (flash photolysis optics C9125). Excitation of the SrSRI
samples was done by 556-nm nanosecond laser pulses (FWHM;
3.5 nm) from a Nd-YAG laser apparatus (LS-2134UT-10;
LOTIS TII: 355 nm, 7 ns, 60 mJ) through an Optical Parametric
Oscillator (OPO) (LT-2214-OPO/PM; LOTIS TII). The repetition rate of the laser pulses was 10 Hz and was reduced to 0.5 Hz
using a beam shutter unit (BD-10MN-OPO; LOTIS TII) and afrequency divider to wait for completion of the photocycle of
SrSRI. The energy of one laser pulse is 3.8 mJ. Synchronization of data acquisition and laser triggering was performed
using a delay pulse generator (DG535; Stanford Research
Systems, Location Sunnyvale, CA). For signal-to-noise
improvement, twenty photoreactions were averaged for one
time slice and 3 sets of time-resolved light-induced difference spectra were recorded and averaged for a sample solution (experiment length of ⬃6 h). Two experiments were
performed and in all, 6 sets of transient spectra were averaged. The bleach of the sample was negligible (⬃5%) in eachexperiment. The temperature of the sample was kept at 20 °C
by circulating water through a thermoregulated bath (NCB1200; EYELA, Tokyo, Japan).
Protein Stability in Low Salt Conditions—Absorption spectra were obtained using a UV-2550 (Shimadzu) spectrometer. To monitor the effects of salt concentration on theabsorbance of SRI, PG-reconstituted samples were resuspended in a buffer containing 50 mM Tris-Cl, pH 7.0. Samples were taken at different time intervals, and the absorption spectra were measured.

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SRI from a Eubacteriumtransformed only by the vector pET21c(⫹), whereas cells containing SRU_2579 did not show any visible color, implying it
was not expressed in the E. coli cells. SRU_2511 is hereafter
called SrSRI. Purified SrSRI was reconstituted into PG liposomes, and the absorption spectrum was obtained as shown in
Fig. 2. The absorption maximum of SrSRI is located at 558 nm.
That maximum wavelength is similar to that of HsSRI (587 nm)(1) and is significantly different from the sensory rhodopsin II
whose maximum is located at around 500 nm (36, 37). Thus, wewere able to express a sensory rhodopsin I-like protein as aphotoactive protein.
Retinal Configuration and pKa of the Counterion—To investigate the physical properties of SrSRI, HPLC analysis and pHtitration experiments were carried out. In the dark, light-driven
ion pumps (BR and HR) use all-trans and 13-cis retinal (38, 39),
whereas the photosensors HsSRI, HsSRII, and NpSRII use only
all-trans retinal (40, 41). Fig. 3 shows the retinal isomer composition. The isomeric state of retinal is predominantly alltrans, with a small proportion of 13-cis, indicating that the retinal composition is markedly sensory rhodopsin-like. In all
retinal proteins (except for Halorhodopsins), the protonated
Schiff base is stabilized by an aspartic acid as a counterion.
Spectroscopic pH-titration was performed to estimate the pKavalue of the counterion of SrSRI (Asp-72) in the unphotolyzed
state because protonation of Asp-72 causes a red-shift at theabsorption maximum (see Fig. 4a). Fig. 4b shows that difference
absorption spectra. Each spectrum was obtained by subtractingthat of pH 8.5. The different spectra clearly showed that the acidform has 2-fold larger extinction coefficient than the alkaline
form as well as HsSRI (42). The titration curves were analyzed

FIGURE 3. Chromophore configuration extracted from SrSRI reconstituted into PG liposomes with 1 M NaCl and 50 mM Tris-Cl (pH 7. 0). The
detection beam was set at 360 nm. Ts, Ta, 13s, and 13a stand for all-trans15-syn retinal oxime, all-trans 15-anti retinal oxime, 13-cis 15-syn retinal
oxime, and 13-cis 15-anti retinal oxime, respectively. The molar compositionof retinal isomers was calculated from the areas of the peaks in the HPLC
patterns. The chromophore configuration extracted from SrSRI suspended inbuffer solution containing 0.1% DM is given in parentheses.

using the Henderson-Hasselbalch equation with a single pKavalue (43) (Fig. 4c). The pKa value of Asp-72 for the PG-reconstituted SrSRI was 4.3, which is 3 units lower than that of HsSRI(⬃7.2) (44), suggesting a structural difference around the chromophore between SrSRI and HsSRI. Although the pKa value of
SrSRI might be affected by interaction with SrHtrI because thevalue of HsSRI is elevated to 8.5 upon HsHtrI association (44),the lower pKa of SrSRI also indicates that Asp-72 exists in a
deprotonated form at neutral pH where the bacteria exist, suggesting the functional importance of the deprotonated state of
SrSRI.
Photocycle of SrSRI as Revealed by Time-resolved Flash Photolysis and Low Temperature UV-Vis Spectroscopy—To confirm the photocycle of SrSRI, flash photolysis and low temperature UV-Vis spectroscopy were used (Figs. 5 and 6), and aschematic drawing of the photocycle based on the results isshown in Fig. 7. The upper panel in Fig. 5 shows the flashinduced difference spectrum of DM-solubilized SrSRI over the
spectral range from 300 to 700 nm. In this experiment, theSrSRI sample contained 0.1% DM as a detergent to increase
the S/N ratio without inactivation. Depletion by the flash andrecovery of the original pigment was observed at 557 nm. The
value was almost the same as the absorption maximum of SrSRI
shown in Fig. 2 (558 nm). At 390 nm, an increase and a decreaseof absorbance were observed, implying the formation and decay

FIGURE 1. a, secondary structure of S. ruber sensory rhodopsin I (SrSRI, SRU_2511). b, alignment of putative amino acid sequences of SRIs reported so far (29)with SrSRI. Names of archaeal and bacterial sensory rhodopsins and sources from top are: cso_Hva, Haloarcula vallismortis cruxsensory rhodopsin (GenBankTM
code: D83748); sop-F5R, H. salinarum strain Flx5R sR (L05603); sop-SGl, Hb. sp. strain SGI sR (X70290); SRU_2511, S. ruber strain DSM 13855 (3852586); SRU_2579,
S. ruber strain DSM 13855 (3852268). Amino acid residues marked by an asterisk in helices F and G are important residues for phototaxis in SRI. The amino acidresidues identified as necessary for function (31) were conserved among them except for a His residue (His-166 in HsSRI).

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FIGURE 2. The absorption spectrum of SrSRI. The protein was reconstituted
into PG liposomes and was resuspended in buffer solution (50 mM Tris-HCl,
pH 7.0, 1 M NaCl). The temperature was kept at 20 °C.

SRI from a Eubacteriumof an intermediate SrSRIM similar to HsSRIM (S373) where an
absorbance maximum is located at 373 nm. The lower panel inFig. 5 shows the time courses of the absorbance change atselected wavelengths (390 nm for the M state and 557 nm forthe unphotolyzed state). The M-decay rate is estimated as 6 s⫺1
by single exponential equation, close to that of HsSRI (0.8 s⫺1)(2) and of NpSRII (0.56 s⫺1) (45). This slow photocycle is particularly important because a key difference between transportand sensory rhodopsins is the much slower kinetics of the photochemical reaction cycle of the sensors (46). The ion-pumping
FIGURE 4. a, absorption spectrum of PG-reconstituted SrSRI at pH 6. 0, 4.7, and3.9. b, difference absorption spectra. Each spectrum was obtained by subtracting that of pH 8.5. c, pH-titration curves of the counterion, Asp-72, in

23538 JOURNAL OF BIOLOGICAL CHEMISTRYSrSRI. The SrSRI sample was suspended in a mixture of six buffers with 1 M
NaCl. The titration curves were analyzed using the Henderson-Hasselbalch
equation with a single pKa value (43). The temperature was kept at 20 °C.

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FIGURE 5. a, flash-induced difference spectra of DM-solubilized SrSRI over aspectral range from 300 to 700 nm. The purified SrSRI was resuspended in 50
mM Tris-HCl, pH 7.0, 0.1% DM, and 1 M NaCl. Curves 1– 8 are spectra of 10 ms (1),50 ms (2), 90 ms (3), 130 ms (4), 210 ms (5), 330 ms (6), 540 ms (7), and 860 ms(8) after the illumination. The temperature was kept at 25 °C. b, flash-induced
kinetic data of SrSRI at 390 nm representing the M-decay and at 556 nm
representing the recovery of the original SrSRI. The kinetic data of BR werereproduced from Ref. (47) to compare the time range with SrSRI. For BR, theM-intermediate and the unphotolyzed state were monitored at 410 and 570
nm, respectively.

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Downloaded from www.jbc.org at NAGOYA UNIV CENTRAL LIBRARY on October 8, 2008of the most important intermediates for the function of retinal proteins, except for HR. Low temperature FTIR and UV-Vis spectroscopy
showed the formation of a small
amount of an L-like intermediate of
SrSRI as judged by the absorption
maximum and by the changes ofC⫽C stretching vibration (data notshown), suggesting the presence ofan L-intermediate in SrSRI. In
HsSRI as well, K, L, and M intermediates appeared during the photocycle as they do in NpSRII (2, 7). The
two difference spectra (dark minus
light and light minus subsequent
1-min light illumination) in Fig. 6c
are mirror images of each other,indicating that the K intermediates
are photoreversible intermediates.
However, the two difference spectra(dark minus light and light minus
subsequent 1-min light illumination) in Fig. 6d are not mirror
images of each other. Upon accumulation of SrSRIM (Fig. 6d, solidline), the SrSRIM forms a P510-likeblue-shifted intermediate (P525),
which is important for negative
phototaxis in HsSRI (1), and theP525 was also trapped at the temperature region between 170 and
FIGURE 6. Low temperature UV-Vis absorption spectra of light-activated SrSRI (solid lines) and SrSRI in
the dark (dotted lines) measured at 77 K (a) and at 200 K (b). The protein was reconstituted into PG lipo- 243 K. In HsSRI, the M intermediatesomes with NaCl. The difference spectra shown in c and d, were calculated from the spectra recorded before (S373) becomes the P510-intermeand after irradiation with light. One division of the y-axis of a, b, c, and d corresponds to 0.1, 0.1, 0.05, and 0.05 diate by illumination with UV lightabsorbance unit, respectively.
(1). We confirmed that the P510-intermediate of HsSRI is also accumurhodopsins (BR, HR, and proteorhodopsin) have been optilated in our condition. The identified photocycle scheme ismized for fast photocycling rates to make them efficient pumps
described in Fig. 7.
(see the gray line in Fig. 5b and Ref. 47). In contrast, the sensory
Protein Stability of SrSRI—In 1982, using a BR mutant fromrhodopsins HsSRI and NpSRII have slow photocycles (2, 45),
the archaeon H. salinarum that was believed to express only HR
which allows the transient accumulation of long-lived signalingat that time, Tsuda et al. (48) and Hazemoto et al. (49), foundstates of the receptors to catalyze a sustained phosphorylation
two independent fast and slow photocycles. Bogomolni andcascade controlling flagellar motor rotation. The slow photoSpudich (50) proposed that the pigment having the slow phocycle of SrSRI therefore is a similar property to that of sensory
tocycle is the receptor for phototaxis in this bacterium, and theyrhodopsins from other sources.
proved their hypothesis by flash photolysis of a mutant lacking
Fig. 6 shows low temperature UV-Vis absorption spectra ofboth BR and HR, which showed almost the same level of pholight-activated SrSRI (solid lines) and SrSRI in the dark (dotted
totaxis as that of the wild type (51). They named this retinallines) measured at 77 K (a) and at 200K (b). The difference
pigment protein sensory rhodopsin I (SRI). Of interest, thespectra shown in the panels (c and d) were calculated from theground state of SRI functions as a sensor for positive phototaxis
spectra recorded before and after irradiation with light. At 77 K,
whose action maximum is located at ⬃580 nm, while the longa light-induced spectral red shift was observed, implying the
lived photointermediate absorbing maximally at 377 nm works
formation of a K-intermediate of SrSRI. The formation of aas sensor for negative phototaxis (1). The signal relay mecha3
K-intermediate was also confirmed by FTIR spectroscopy. At
nism from SR receptors to their cognate Htr transducers has
200 K, a spectral blue shift was observed, implying the formabecome a focus of interest, in part because of its importance totion of the M-intermediate of SrSRI. The M-intermediate is one
the general understanding of communication between integral
membrane proteins, about which little is known. Important3
advances in understanding the molecular mechanism of negaY. Sudo, unpublished data.

SRI from a Eubacterium

Acknowledgments—We thank Akira Kawanabe for technicalassistance in the HPLC experiments, and we are also very gratefulto Dr. Mike Dyall-Smith and Dr. David Burns for the gift of thestrain S. ruber.
REFERENCESFIGURE 8. Stability of HsSRI and SrSRIin the absence of NaCl (solid lines)
and 50 mM NaCl (dotted lines). The time-dependent absorbances of theproteins were plotted. The samples were reconstituted into PG-liposomes
and resuspended in a buffer containing 50 mM Tris-Cl (pH 7.0), and the temperature was maintained at 20 °C.

tive phototaxis regulated by the SRII-HtrII complex has been
made in recent years because NpSRII is much more stable than
HsSRI, especially in dilute salt solutions. Therefore, little is
known about the molecular mechanism for positive phototaxis.
In fact, the high-resolution crystal structures of BR, HR, andSRII and the SRII-HtrII complex were reported from 1997
through 2006 (52–56), whereas the structure of SRI has notbeen determined yet. Fig. 8 shows the protein stabilities of
HsSRI and SrSRI under low salt conditions (0 and 50 mM NaCl)as judged by the absorbance of the proteins. A time-dependent
decrease in the absorbance of HsSRI was observed, implying the23540 JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 7. Schematic of the photochemical reaction cycle of SrSRI identified in this study. h␷ indicates light-dependent reactions, and the otherarrows represent thermal reactions. The decay rate constant of SrSRIM (6 s⫺1)
was obtained by a single exponential equation by the flash photolysis experiments shown in Fig. 5. HsSRI absorbs orange light and forms the long-lived
S373 intermediate, which forms the P510 intermediate upon the second photon absorption in the near-UV region (2).

degeneration of the protein within 5 h, whereas SrSRI maintained its spectral stability over the 24 h (Fig. 8). As described in
the pH-titration experiment, SrSRI is also stable over a wide pH
range (pH 3.5– 8.5) (Fig. 4). In addition, PG-reconstituted SrSRI
is stable even in the absence of NaCl, over a wide pH range (pH
2.5–10) and at high temperature (⬃60 °C) (data not shown),
while HsSRI is not stable under those conditions. Thus, SrSRI
expressed in E. coli shows high stability, making it possible toprepare large amounts of protein and enabling the study ofmutant proteins, which will open new avenues to investigate
the photosignaling process in the SRI-HtrI system. In the
future, we will examine whether SrSRI and SrHtrI are expressedin native cells, and whether they regulate positive and/or negative phototaxis.
Concluding Remarks—In this study, we present the first characterization of SRI from a eubacterium S. ruber. The SrSRI was
expressed well (5 mg/liter culture) as a recombinant protein
having all-trans retinal as a chromophore, and it is much morestable than SRI from the archaeon H. salinarum (HsSRI) undervarious conditions. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of SRI fromthe archaeon H. salinarum. In the future, we will examine
whether SrSRI and SrHtrI regulate positive and/or negative
phototaxis.

SRI from a Eubacterium

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