12750

Biochemistry 2008, 47, 12750–12759

Structural Changes of Salinibacter Sensory Rhodopsin I upon Formation of the K
and M Photointermediates†
Daisuke Suzuki,‡ Yuki Sudo,‡ Yuji Furutani,§ Hazuki Takahashi,§ Michio Homma,‡ and Hideki Kandori*,§
DiVision of Biological Science, Graduate School of Science, Nagoya UniVersity, Chikusa-ku, Nagoya, 464-8602, Japan, andDepartment of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, 466-8555, Japan
ReceiVed July 19, 2008; ReVised Manuscript ReceiVed September 23, 2008

ABSTRACT: Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature

because of its ability to mediate opposite signals depending on light color by photochromic one-photon
and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI).
This protein allows more detailed information about the structure and structural changes of SRI during its
action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI,
and the spectral changes upon formation of the K and M intermediates are compared with those of otherarchaeal rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory rhodopsin II (SRII),
bacteriorhodopsin (BR), and halorhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP)
vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion
takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff baseregion in BR and HR. It appears that sensor and pump functions are distinguishable from the spectralfeature of HOOP modes. The HOOP band at 864 cm-1 in SRII, important for negative phototaxis, isabsent in SrSRI, suggesting differences in signal transfer mechanism between SRI and SRII. The stronglyhydrogen-bound water molecule, important for proton pumps, is observed at 2172 cm-1 in SrSRI, as wellas in BR and SRII. The formation of the M intermediate accompanies the appearance of peaks at 1753(+) and 1743 (-) cm-1, which can be interpreted as the protonation signal of the counterion (Asp72) andthe proton release signal from an unidentified carboxylic acid, respectively. The structure and structuralchanges of SrSRI are discussed on the basis of the present infrared spectral comparisons with otherrhodopsins.

Microorganisms respond and adapt to various environmental stimuli. They show attractant and repellent responses(taxis) to survive in various environments where they areliving. Taxis to chemicals including to some amino acids,
temperature, pH, and light is known as chemotaxis, thermotaxis, pH-taxis, and phototaxis, respectively (1). The
archaeon Halobacterium salinarum has two photoreceptors
regulating negative and positive phototaxis. One of them,sensory rhodopsin I (SRI)1works as a dual receptor both fornegative and positive phototaxis (2). Sensory rhodopsin II(SRII, also known as phoborhodopsin, pR) is a negative
phototaxis receptor in the cells (3). SRI and SRII form 2:2
complexes with their transducer proteins, halobacterial†
This work was supported by grants from Japanese Ministry of
Education, Culture, Sports, Science, and Technology to Y.S. (No.
20050012), to Y.F. (No. 19045015), and to H.K. (Nos. 19370067 and20050015).
* To whom correspondence should be addressed. Phone and Fax:
81-52-735-5207. E-mail: kandori@nitech.ac.jp.
‡
Nagoya University.
§
Nagoya Institute of Technology.
1
Abbreviations: SrSRI, sensory rhodopsin I from Salinibacter ruber;
FTIR, Fourier transform infrared; SrSRIK, K-intermediate of SrSRI;
SrSRIM, M-intermediate of SrSRI; HsSRI, sensory rhodopsin I fromHalobacterium salinarum; BR, light-adapted bacteriorhodopsin fromHalobacterium salinarum; HR; halorodopsin from Natronomonas
pharaonis; SRII, sensory rhodopsin II from Natronomonas pharaonis;
BR-T, P200T/V210Y/A215T triple mutant of BR; HOOP, hydrogen
out-of-plane.

transducer protein I for SRI (HtrI) and halobacterial transducer protein II for SRII (HtrII) (4, 5), and transmit lightsignals to a two-component signal transduction cascade,which controls the flagella motor rotation (6).
SRI is one of the most interesting photosensory receptors
in nature because of its novel ability to mediate opposite
signals depending on light color by photochromic one- andtwo-photon reactions (2, 7). However, the molecular mechanism of SRI is not understood in atomic terms. On the other
hand, the signal relay mechanism of SRII is well characterized by using various methods because SRII from Natronomonas pharaonis (NpSRII) has high stability in dilute salt
solutions and is much more resistant against detergents (8, 9),whereas HsSRI is unstable in those conditions. Recently, wecharacterized an extremely stable SRI-like protein from aeubacterium Salinibacter ruber, which was named SrSRI(10). This protein is the first eubacterial SRI as a functional
protein. SrSRI has an all-trans retinal as a chromophore with
an absorption maximum at 558 nm and shows a slow
photocycle. These properties are close to those of HsSRI.
SrSRI is highly stable within the membrane and in detergent
micelles even in the absence of NaCl. In addition, afunctional expression system utilizing Escherichia coli cells
can provide a large amount of the proteins (10).
Besides SRI and SRII, H. salinarum has two other retinal
proteins, bacteriorhodopsin (BR) and halorhodopsin (HR),10.1021/bi801358b CCC: $40.75  2008 American Chemical Society
Published on Web 11/08/2008

FTIR spectroscopy of SrSRIFIGURE 1: Predicted secondary structure of Salinibacter ruber
sensory rhodopsin I (SrSRI). Names of the other rhodopsins are
HsSRI, sensory rhodopsin I from Halobacterium salinarum; BR,
bacteriorhodopsin from Halobacterium salinarum; NpHR, halorhodopsin from Natronomonas pharaonis; and NpSRII, sensory
rhodopsin II from Natronomonas pharaonis. Amino acid residues
colored with yellow are necessary for function in HsSRI. The
N165F, H166Y, and D201N mutations convert the normally
attractant signal to a repellent signal (7, 60, 61). The R215W is a
suppressor mutation (62). We focused on the residues colored pink.
A strongly hydrogen-bonded water molecule is shown as a filledgreen circle.

which work as light-driven ion pumps, whose functions are
distinctly different from those of SRI and SRII. Namely, BR
and HR function as a light-driven outward proton pump anda light-driven inward chloride pump, respectively. The
original state of SRI (λmax ) 587 nm) and its long-lived
photointermediate (the M-intermediate; λmax ) 373 nm) are
important for positive and negative phototaxis, respectively(6). SRII is a negative phototaxis receptor in haloarchaea,
including H. salinarum and N. pharaonis. Those are called
HsSRII and NpSRII, respectively, and their absorption
maxima are at about 500 nm (8). Thus, haloarchaea are
attracted to light with wavelengths longer than 520 nm, andthey avoid light with wavelengths shorter than 520 nm dueto the functions of SRI and SRII (6). Light at >520 nm canactivate the ion pumping rhodopsins (BR and HR) to obtainlight-energy, and cells avoid light of shorter wavelengths,
which contains harmful near-UV. Interestingly it was reported that SRI and SRII are able to function as an outward
proton pump like BR only in the absence of the cognate
transducer proteins (11, 12), suggesting that these retinal
proteins mentioned above are presumably evolved from thesame protein and have similar structures (Figure 1). In fact,
the rhodopsins have seven transmembrene helices and aretinal chromophore bound to a specific lysine residue ofhelix G via a protonated Schiff base linkage (13). To stabilize
the positive charge of a protonated Schiff base inside theprotein, highly conserved charged groups are present in the
Schiff base region: arginine and aspartate of helix C andaspartate of helix G. Water molecules also stabilize thecharged group in the region (13). In particular, three watermolecules are involved in the pentagonal cluster structure

Biochemistry, Vol. 47, No. 48, 2008 12751(14, 15). A strongly hydrogen-bound water molecule locatedbetween the Schiff base and the counterion exists in the lightdriven proton pumps, BR (16), transducer-free SRII (17),HR with azide (18), and D212N BR mutant with NaCl (19).
Thr204 in SRII located around the retinal chromophore (Thedistance from the C14 atom to the hydroxyl oxygen of
Thr204 is 4.4 Å.) is quite an important residue for negative
phototaxis (15, 20). Thus, structural changes of the Schiffbase region and internal water molecules are directly linked
to functions of retinal proteins.
Light absorption of these retinal proteins triggers a cyclic
reaction that is comprised of a series of intermediates,
designated alphabetically (21). The trans-cis photoisomerization of the retinal chromophore leads to the formation of
the K intermediate. In proton pumps, the primary protontransfer takes place in the L to M transition. A proton istransferred from the protonated Schiff base (Lys205 for
HsSRI; Lys216 for BR; Lys205 for SRII) to an aspartate ofhelix C (Asp76 for HsSRI; Asp85 for BR; Asp75 for SRII).
In BR, a proton is released simultaneously from a protonatedwater cluster to the extracellular side (22, 23). Consequently
the proton was transferred from the cytoplasmic side to theextracellular side. Thus, the molecular properties of ionpumping rhodopsins, especially BR, have been extensively
studied.
How about photosensory rhodopsins? SRII has been wellcharacterized over the past several years using various
methods because of its high protein stability (24). The
crystallographic structure of SRII and the SRII/HtrII complex
had been achieved (5, 15), and light-induced structuralchanges were also identified by various methods, including
UV-vis, electron paramagnetic resonance (EPR) spectroscopy, etc. Interestingly, the structure and structural changes
are almost the same as those of BR, implying that thefunctional differentiation was caused by small structuraldifferences in some amino acid side chain(s) or main
chain(s). In fact, Sudo and Spudich recently reported that
substitution of only three residues, P200T/V210Y/A215T,
converted BR into a sensor for negative phototaxis like SRII
(BR-T), indicating similar structural changes between BR
and SRII (25). On the other hand, little is known about themolecular mechanism(s) of interactions between SRI and
HtrI, about structural changes, or about the signal relay
mechanism of positive phototaxis.
Here, we utilized Fourier transform infrared (FTIR)
spectroscopy to explore structural changes of SrSRI. FTIR
spectroscopy is a powerful tool to investigate structurefunction relationship in rhodopsins. In the case of SRII, atransducer-dependent specific interaction of Thr204 was firstfound (26), followed by phototaxis analysis revealing theimportant role of Thr204 in function (20). Then, the origin
of such a specific interaction related to Thr204 was revealed
from a positive correlation between the increment of a retinal
vibration in the K intermediate and the physiologicalphototaxis response (27, 28). In HsSRI, earlier studies
identified characteristic vibrational bands such as Asn53 (29)and Asp76 (30). It was also shown that HsSRIM contains
13-cis retinal (31). Hydrated film samples of SrSRI werestable in the absence of salt, in contrast to HsSRI (32). This
enabled us to obtain accurate light-induced difference spectra
upon formation of the K and M intermediates in the entire
mid-infrared region. In this study, we report light-induced

12752Biochemistry, Vol. 47, No. 48, 2008

Suzuki et al.

difference FTIR spectra at neutral pH, where the Schiff basecounterion (Asp72) is deprotonated. The obtained spectra
for SrSRI are compared with those of other archaeal
rhodopsins such as BR, HR, HsSRI, and SRII.
MATERIALS AND METHODS
Preparation of SrSRI and HsSRI Samples. SrSRI andHsSRI were prepared as described previously (10, 32, 33).
Briefly, the SrSRI and HsSRI proteins with a six-histidine
tag at the C-terminus were expressed in E. coli BL21(DE3)cells, solubilized with 1.0% n-dodecyl β-D-maltoside (DDM),
and purified with a Ni2+-NTA column (QIAGEN, Valencia,
CA, USA) as described previously (34). Purified samples
were then reconstituted into L-R-phosphatidylglycerol (PG)
liposomes (SRI/PG ) 1:50 molar ratio) by removing thedetergent with Bio-Beads (SM2, Bio-Rad, Hercules, CA).
FTIR Spectroscopy. Low-temperature FTIR spectroscopy
was performed with 2-cm-1 resolution as described previously (17). The SrSRI samples in PG liposomes were washedwith a 2 mM phosphate buffer (pH 7.0). Then a 90 µL aliquotof the sample (0.1-1 mg) was dried on a BaF2 window witha diameter of 18 mm. The HsSRI samples in PG liposomes
were washed with a 15 mM borate buffer (pH 8.3) containing
300 mM NaCl. Then a 40 µL aliquot of the sample (0.1-1mg) was dried on a BaF2 window with a diameter of 18mm, and deposition of excess salt was washed away with a
15 mM borate buffer (pH 8.3) containing 15 mM NaCl. Afterhydration with H2O, D2O, or D218O, the sample was placedin a cell, which was mounted in an Oxford DN-1704 cryostat
equipped in the Bio-Rad FTS-40 spectrometer.
The SrSRIK minus SrSRI difference spectra were measured
at 77 K as follows. Illumination of the SrSRI film with a
450 nm light for 2 min at pH 7.0 and 77 K converted SrSRI
to SrSRIK, and subsequent illumination with >640 nm lightforced SrSRIK to revert to SrSRI. The SrSRIM minus SrSRI
difference spectra were measured at 260 K and pH 7.0 as
follows. To convert SrSRI to SrSRIM, the sample wasirradiated with >480 nm light for 2 min, after which
subsequent 1 min illumination with UV light changed SrSRIM
back to SrSRI. The difference spectrum was calculated fromthe spectra constructed with 128 interferograms before andafter the illumination. Twenty-four or sixteen spectra obtained in this way were averaged for the SrSRIK minus SrSRIor SrSRIM minus SrSRI spectra. The HsSRIK minus HsSRI
difference spectra were measured at 77 K. Illumination of
the HsSRI film with 500 nm light for 2 min at pH 7.0 and
77 K converted SrSRI to SrSRIK, and subsequent 1 min
illumination with >680 nm light forced HsSRIK to revert to
HsSRI.
BRK minus BR, HRK minus HR, SRIIK minus SRII, BRTK minus BR-T spectra, BRM minus BR, HsSRIM minus
HsSRI, and SRIIM minus SRII spectra were taken from
Shibata et al. (35), Shibata et al. (36), Kandori et al. (34),
Sudo et al. (37), Tanimoto et al. (38), Furutani et al. (32),and Furutani et al. (39), respectively. All spectra werenormalized with respect to the C-C or CdC stretchingvibration of the retinal chromophore.
RESULTSInfrared Spectral Changes of SrSRI and HsSRI upon
Formation of K Photointermediate. Figure 2a,b shows theK minus initial state difference FTIR spectra of SrSRI and

FIGURE 2: SrSRIK minus SrSRI (a) and HsSRIK minus HsSRI (b)difference infrared spectra measured at 77 K at pH 7.0 and 8.5,
respectively, in the 1800-800 cm-1 region. The spectrum of HsSRI
is deleted at <872 cm-1. The HsSRIK minus HsSRI spectrum (b)is multiplied by 4.2 for sake of comparison. The samples were
hydrated with H2O. The SRIIK minus SRII difference FTIR
spectrum (c) is reproduced from ref 34 for comparison. One divisionof y axis corresponds to 0.018 absorbance units.

HsSRI, respectively. The SRIIK minus SRII (Figure 2c) isreproduced from ref 34 for comparison. The spectrum of
HsSRI was too noisy at <872 cm-1 because of scattering,so that the region was removed. The bands at 1527 (-)/
1516 (+) cm-1 for SrSRI, 1529 (-)/1518 (+) cm-1 for
HsSRI, and 1554 (-)/1544 (+) cm-1 for SRII correspondto the ethylenic CdC stretching vibrations of the retinal
chromophore. The lower frequency shift corresponds to thespectral red-shift upon formation of the K intermediate. The
bands at 1250 (-), 1200 (-), 1192 (+), and 1165 (-) cm-1
for SrSRI, 1249 (-), 1199 (-), 1191 (+), and 1163 (-) cm-1
for HsSRI, and 1244 (-), 1203 (-), and 1198 (+) cm-1 for
SRII correspond to the C-C stretching vibrations of theretinal chromophore, and the frequency shift represents
retinal isomerization from the all-trans to 13-cis form. Thus,
these results indicate the formation of the K intermediate
both in SrSRI and HsSRI as well as in SRII. Negative bands
were observed at 1165 cm-1 for SrSRI and 1163 cm-1 for
HsSRI, but not for SRII. It is likely that the bands originate
from C10-C11 stretching vibration, because the corresponding band of BR appears at 1170 cm-1 and is assigned to
C10-C11 stretching vibration (40).
Figure 3 shows the spectral changes in the 1780-1560
cm-1 region, where most signals originate from vibrationsof protein. One exception is the CdN stretching vibrationof the retinal Schiff base. Among various D2O sensitive
bands of SRII (c), the CdN stretch has been assigned at
1650 cm-1 in H2O and 1633 cm-1 in D2O (34). The 1646
and 1622 cm-1 bands for SrSRI are tentatively assigned to
the CdN stretches of the retinal Schiff base in H2O and D2O,

FTIR spectroscopy of SrSRIFIGURE 3: SrSRIK minus SrSRI (a) and HsSRIK minus HsSRI (b)difference infrared spectra measured at pH 7.0 and 8.5, respectively,in the 1780-1565 cm-1 region. The HsSRIK minus HsSRI spectra
are multiplied by 4.2 for sake of comparison. The samples were
hydrated with H2O (s) or D2O ( · · · ). Underlined values correspondto CdN stretching vibration. The SRIIK minus SRII difference FTIR
spectra (c) are reproduced from ref 34 for comparison. One divisionof y axis corresponds to 0.008 absorbance units.

respectively. Previous resonance Raman of HsSRI reported
those frequencies at 1634 and 1616 cm-1 (31). Therefore,
the 1641 and 1624 cm-1 bands in Figure 3b probably
originate from the Schiff base CdN stretches of HsSRI in
H2O and D2O, respectively. The spectral upshift in H2O iscaused by the coupling of the N-H bending vibration of
the Schiff base, and the difference in frequency between H2O
and D2O has been regarded as the marker of hydrogenbonding strength of the Schiff base nitrogen. Frequency
differences in SrSRI (1646 - 1622 ) 24 cm-1) and HsSRI(1641 - 1624 ) 17 cm-1) are similar to the value in SRII
(1650 - 1633 ) 17 cm-1), indicating that the hydrogenbonding strengths of the Schiff base in SRIs are similar tothat of SRII. In the K state, the CdN stretching vibrations
appear at 1610-1600 cm-1, though the band in D2O isunclear only for HsSRI (Figure 3b).
In SRII, the 1704 (-)/1700 (+) cm-1 bands were assigned
as the CdO stretching vibration of Asn105, and the strong
peaks are characteristic for SRII (41). Corresponding aminoacids are Asp102 in SrSRI and Asp106 in HsSRI. In SrSRIand HsSRI, the 1744 (-)/1737 (+) and 1740 (-)/1734 (+)
cm-1 bands in H2O shifted to 1734 (-)/1730 (+) and 1733(-)/1726 (+) cm-1 in D2O (Figure 2a,b), respectively. They
were ascribable to the CdO stretching vibrations of Asp102
for SrSRI and Asp106 in HsSRI, respectively. The corresponding bands appear at 1742 (-)/1733 (+) cm-1 in BR
and 1744 (-)/1737 (+) cm-1 in HR (41-43), and theirintensities are similar to those of SrSRI and HsSRI. It should
be noted that the carboxylic CdO stretch of the N105D
mutant SRII exhibits intense bands at 1744(-)/1739(+) cm-1

Biochemistry, Vol. 47, No. 48, 2008 12753

FIGURE 4: SrSRIK minus SrSRI (a) and HsSRIK minus HsSRI (b),difference infrared spectra measured at pH 7.0 and 8.5, respectively,in the 1080-780 cm-1 region. The spectrum of HsSRI was deleted
at <872 cm-1. The HsSRIK minus HsSRI spectra are multipliedby 4.2 for sake of comparison. The samples were hydrated with
H2O (s) or D2O ( · · · ). The SRIIK minus SRII (c), BR-TK minus
BR-T (d), BRK minus BR (e), and HRK minus HR (f) difference
FTIR spectra are reproduced from refs 34, 37, 35, and 36,
respectively, for comparison. One division of y axis corresponds
to 0.006 absorbance units.

in H2O and at 1734(-)/1728(+) cm-1 in D2O (41). Thus,
the structure and structural changes at position 102 in SrSRI(106 in HsSRI) are similar to those of ion-pumping rhodopsins.
Figure 4 shows spectral changes in the 1080-780 cm-1
region, where hydrogen out-of-plane (HOOP) vibrations of
the retinal molecule are mainly observed. Difference FTIR
spectra for SRII (c), BR-T (d), BR (e), and HR (f) are
reproduced from refs 34, 37, 35, and 36, respectively, forcomparison. The SrSRIK minus SrSRI and the HsSRIK minus
HsSRI spectra exhibit intense positive peaks at 998, 984,
977, 963, and 956 cm-1 for SrSRI, and at 985, 977, 964,
and 956 cm-1 for HsSRI, which would be tentatively
assigned to HOOP vibrations of the retinal chromophore.
These results suggest that the structural changes spread tothe middle part of the retinal chromophore in SRI. By using
all seven monodeutrated retinal analogues, Furutani et al.
assigned the 966 (+)/971 (-) and 958 (+)/961 (-) cm-1
bands in SRII as the C7dC8 and C11dC12 Au (irreducible
representation of diene vibrations) HOOP modes, respectively, and the positive bands at 1001, 994, 987, and 979
cm-1 as the C15-HOOP vibrations (40). From the similarity
in frequency, the bands at 998, 984, and 977 cm-1 in SrSRI
are likely to originate from C15-HOOP (Figure 4a). The
bands at 963 and 956 cm-1 are likely to originate from
C7dC8 and C11dC12 Au HOOP vibration, respectively.
These bands were also observed in a BR mutant engineered
to transmit photosensory signals like SRII (BR-T) at 964(+)/971 (-), 958 (+)/961 (-), 996 (+), 992 (+), and 979

12754

Biochemistry, Vol. 47, No. 48, 2008(+) cm-1 (Figure 4d), implying the importance of theextended chromophore distortion for phototaxis signaling.
In addition, a positive band at 1012 cm-1 appears both in
SrSRIK and HsSRIK. A similar band is also present at 1024
cm-1 in SRIIK and at 1023 cm-1 in BR-TK, which was
assigned to symmetric rocking of methyl groups connectedto the C9 and C13 atoms (37, 40).
On the other hand, only two intense positive peaks wereobserved for ion-pumping rhodopsins, at 974 and 957 cm-1
for BR (Figure 4e) and at 993 and 968 cm-1 for HR (f). For
the BR spectrum (e), the D2O-sensitive intense bands at 974(+) and 957 (+) cm-1 were assigned as HOOP vibrationsof C15-H and N-H groups (44), indicating that thestructural changes of BR upon formation of the K intermediate are localized to the Schiff base region unlike photosensory
rhodopsins. Thus, the HOOP modes were highly different
between photosensors (Figure 4a-d) and ion-pumps (e, f).
The bands at 864 and 861 cm-1 were assigned as C14HOOP in SRII (Figure 4c) and BR-T (d), respectively, thepresence of which is highly correlated with negative phototaxis (28). It appears that SrSRI does not possess such aband (Figure 4a). Instead, SrSRI possesses a peak at 803
cm-1, which was absent for SRII and BR-T but present for
BR. In the BR spectra (Figure 4e), two intense positive bands
at 811 and 803 cm-1 are likely to originate from wagging
vibrations of the retinal chromophore, because resonanceRaman study reported that such vibrations appeared in the
900-800 cm-1 region (45). Therefore the same band in
SrSRI would originate from the vibration, although the bandat 803 cm-1 is more largely shifted upon D2O hydration thanthose in BR.
Figure 5 shows the spectral changes in D2O in the2560-1900 cm-1 region, which contains O-D and N-D
stretching vibrations. The measurements for SRII, BR, andHR are reproduced from the published spectra (34-36). The
bands at 2515 (-)/2474 (+) cm-1 of SRII (b) and 2506 (-)/
2466 (+) cm-1 of BR (c) were previously assigned as the
O-D stretch of Thr79 and Thr89, respectively (46, 47).
Therefore, similar bands at 2526 (-)/2471 (+) cm-1 of SrSRI(a) that show no isotope shift of D218O possibly originate
from the O-D stretch of a conserved threonine residue(Thr76) (Figure 1). A low frequency (2526 cm-1) as
threonine O-D stretch is an indication of a strong hydrogenbond of Thr76 in SrSRI, presumably with a counterion,
Asp72. A lower frequency shift from 2526 to 2471 cm-1
implies a further strengthened hydrogen bond of Thr76 uponretinal photoisomerization.
Some peaks in this frequency region exhibit spectral
downshift by ∼10 cm-1 in D218O (blue spectra in Figure 5),
which originate from O-D stretching vibrations of watermolecules. Proton-pumping rhodopsins possess stronglyhydrogen-bonded water molecule(s), whose O-D stretch islocated at <2400 cm-1 (48). The water stretches of SRII at
2307 and 2215 cm-1 (Figure 5b) and those of BR at 2323,
2292, and 2171 cm-1 (Figure 5c) correspond to stronglyhydrogen-bonded water molecules (16, 17). HR has only
moderately hydrogen-bonded water molecules at 2535, 2501,
and 2445 cm-1 in the unphotolyzed state, though retinal
isomerization strengthens the water hydrogen bond as shownat 2384 and 2263 cm-1 (Figure 5d) (36). In the case of SrSRI,
a strongly hydrogen-bonded water molecule was observed
at 2172 cm-1 (Figure 5a). In addition, water bands were

Suzuki et al.

FIGURE 5: SrSRIK minus SrSRI (a) difference infrared spectra
measured at pH 7.0 in the 2560-1900 cm-1 region. The samples
were hydrated with D2O (red line) and with D218O (blue line).
Underline, green label, and purple label correspond to CdN
stretching vibration, vibration of water molecules, and vibration of
threonine residue, respectively. The SRIIK minus SRII (b), BRK
minus BR (c), and HRK minus HR (d) difference FTIR spectra are
reproduced from refs 34, 35, and 36, respectively, for comparison.
One division of y axis corresponds to 0.0012 absorbance units.

observed at 2434 (-)/2368 (+) cm-1 (Figure 5a), as well as
2651 and 2611 cm-1 (data not shown).
The peaks at 2063 and 2037 cm-1 in SrSRI (Figure 5a)do not originate from the water O-D stretch. The peaks at
2140 and 2091 cm-1 in SRII (b) and those at 2173 and 2123
cm-1 in BR (c) were previously assigned to the N-D
stretches of the Schiff base, using 15N-Lys containing
proteins (47, 49). Therefore, the 2063 and 2037 cm-1 bandsin SrSRI are good candidates for the Schiff base vibration.
Lower frequencies of these bands indicate that hydrogenbonding strength of the N-D group in SrSRI is stronger
than those of BR and SRII, which is consistent with theanalysis of CdN stretching vibrations (Figure 3) (34, 35).
Infrared Spectral Changes of SrSRI, HsSRI, and SRII upon
Formation of M Photointermediate. Figure 6a shows the M
minus initial state difference FTIR spectra of SrSRI (a).
Difference FTIR spectra for HsSRI (b) and SRII (c) are
reproduced from refs 32 and 39, respectively, for comparison.
The bands at 1525 (-)/1562 (+) cm-1 for SrSRI, 1525 (-)/
1559 (+) cm-1 for HsSRI, and 1545 (-)/1568 (+) cm-1 for
SRII correspond to the ethylenic CdC stretching vibrationsof the retinal chromophore, but amide-II vibrations also
possibly appear. In fact, resonance Raman data reported the
CdC stretches of the M state of HsSRI at 1568 cm-1 (31),
not at 1559 cm-1. Negative bands at 1249, 1199, and 1164
cm-1 for SrSRI are attributable to the C-C stretchingvibration of the retinal chromophore, where intensity of thecorresponding positive bands is much reduced because of

FTIR spectroscopy of SrSRIFIGURE 6: SrSRIM minus SrSRI (a) difference infrared spectra
measured at 260 K at pH 7.0 in the 1800-800 cm-1 region. The
samples were hydrated with H2O. The HsSRIM minus HsSRI (b)and SRIIM minus SRII (c) difference FTIR spectra are reproduced
from refs 32 and 39, respectively, for comparison. One division ofy axis corresponds to 0.01 absorbance units.

deprotonation of the Schiff base. All three spectra show apositive peak at 1397-1393 cm-1. In addition, the SrSRIM
minus SrSRI spectrum shows an additional peak at 1414
cm-1 (Figure 6a). Since COO- stretching vibrations ofcarboxylate appear in this frequency region, the positive bandmay correspond to deprotonation of a protonated carboxylic
acid at 1743 cm-1.
Figure 7 shows the spectral changes in the 1780-1600
cm-1 region. When the Schiff base counterion (Asp72 for
SrSRI; Asp76 for HsSRI; Asp75 for SRII) is deprotonated,
the M formation accompanies a proton transfer from the
Schiff base to the counterion. The experimental conditions
are indeed the cause, because the pH is higher than the pKaof the counterion. In SRII, the positive band at 1765 cm-1
in H2O and at 1753 cm-1 in D2O represents the protonation
signal of Asp75 (Figure 7c) (39). In HsSRI, the positive bandat 1749 cm-1 band in H2O and at 1739 cm-1 in D2O wereassigned as the CdO stretching vibration of Asp76 (Figure7b) (30, 32). In the case of SrSRI, we observed a positive
band at 1753 cm-1 in H2O, which shifts to 1742 cm-1 in
D2O (Figure 7a). Therefore, it is reasonable to assign theband as the CdO stretching vibration of Asp72.
Interestingly, the SrSRIM minus SrSRI spectrum exhibits
an additional negative band at 1743 cm-1 in H2O, shifting
to 1734 cm-1 in D2O. At present, we cannot exclude thepossibility that the bands at 1753 (+)/1743 (-) cm-1
originate from a carboxylic acid (hydrogen-bonding alterationof a protonated carboxylic acid), but it is unlikely because
the M formation probably accompanies a proton transfer toAsp72. Thus, a protonated carboxylic acid undergoes deprotonation upon formation of the M intermediate. A positive
band at 1414 cm-1 (Figure 6a) possibly corresponds to the

Biochemistry, Vol. 47, No. 48, 2008 12755

FIGURE 7: SrSRIM minus SrSRI (a) difference infrared spectra
measured at 260 K at pH 7.0 in the 1780-1600 cm-1 region. The
samples were hydrated with H2O (s) or D2O ( · · · ). Underlined
values corresponds to CdN stretching vibration. TheHsSRIM minus
HsSRI (b) and SRIIM minus SRII (c) difference FTIR spectra are
reproduced from refs 32 and 39, respectively, for comparison. One
division of y axis corresponds to 0.01 absorbance units.

signal of the carboxylate deprotonation. In BR, the Mformation accompanies proton release from a protonatedwater cluster, not from a carboxylic acid (22, 23). The present
observation may indicate proton release from a carboxylic
acid at the extracellular side of SrSRI, which is also the casein some BR mutants (50).
Figure 7 shows the CdN stretching vibrations in H2O
and D2O: at 1644 and 1624 cm-1 in SrSRI (a), at 1640
and 1626 cm-1 in HsSRI (b), and at 1655 and 1632 cm-1
in SRII (c), respectively. On the other hand, H/Dinsensitive bands can be identified as an amide-I vibration.
Figure 7 shows such bands at 1666 (+)/1654 (-) cm-1
for SrSRI (a), at 1661 (+)/1647 (-) cm-1 for HsSRI (b),and at 1663 (-)/1644 (+) cm-1 for SRII (c), whichcorrespond to an amide-I vibration of the R-helix. Interestingly, frequency shifts were opposite between SRI and
SRII, where a downshift and an upshift were observed,
respecively. This indicates that the M formation accompanies a weakened hydrogen bond of the R-helix in
SRI but a strengthened hydrogen bond of the R-helix in
SRII. In other words, activation of SRI or SRII may
involve breakage or formation of the helical structure.
Figure 8 shows the spectral changes in D2O in the2740-1900 cm-1 region. Difference FTIR spectra for SRII(b) and BR (c) are reproduced from refs 39 and 38,
respectively, for comparison. The bands at 2704 (+), 2690(-), and 2640 (+) cm-1 in the SrSRIM minus SrSRI spectrum(Figure 8a) can be identified as water O-D stretchingvibrations, whereas no isotope effect of water molecules wasobserved at <2500 cm-1. Absence of the negative 2172 cm-1
band (Figure 5a) in Figure 8a indicates that the strongly

12756

Biochemistry, Vol. 47, No. 48, 2008

FIGURE 8: SrSRIM minus SrSRI (a) difference infrared spectra
measured at 260 K at pH 7.0 in the 2760-1900 cm-1 region. The
samples were hydrated with D2O (red line) and with D218O (blueline). Underline, green label, and purple label correspond to CdN
stretching vibration, vibration of water molecules, and vibration of
threonine residue, respectively. The SRIIM minus SRII (b) and BRM
minus BR (c) difference FTIR spectra are reproduced from refs 39
and 38, respectively, for comparison. One division of y axis
corresponds to 0.0012 absorbance units.

hydrogen-bonded water molecule in SrSRI restores its
hydrogen-bonding strength upon formation of the M intermediate. This is also the case for strongly hydrogen-bonded
water molecules in BR (2323, 2292, and 2171 cm-1 bands
in Figure 5c) and SRII (2215 cm-1 band in Figure 5b), but
only the negative water band at 2307 cm-1 in SRII changes
its hydrogen bond in SRIIM, presumably shifting to 2416
cm-1 (Figure 8b).
The negative bands at 2148 and 2090 cm-1 in SRII
(Figure 8b) and at 2164 and 2118 cm-1 in BR (c) wereassigned to the N-D stretch of the Schiff base (38, 51).
Similar bands were observed at 2074 and 2030 cm-1 in
SrSRI (Figure 8a), whose frequencies are similar to thosein the SrSRIK minus SrSRI spectrum (Figure 5a). Therefore it is likely that the bands originate from the N-D
stretch of the Schiff base. There are also bands at 2504(-) cm-1 in SRII (Figure 8b) and at 2506 (-) and 2466(+) cm-1 in BR (c), which were assigned as the O-D
stretch of Thr79 and Thr89, respectively (46, 47). Similarly, the bands at 2490 (-) and 2396 (+) cm-1 in SrSRI
could originate from the O-D stretch of Thr76.
Figure 9 shows the infrared spectra of the SrSRIK minus
SrSRI (a) and SrSRIM minus SrSRI (b) in D2O in the3600-3000 cm-1 region, where H/D unexchangeable O-H
and N-H stretching vibrations appear. The candidates are
N-H stretching vibrations of the peptide backbone (amideA) and O-H or N-H stretching vibrations of threonine,tyrosine, and tryptophan. In the case of SRII, transducerdependent bands were observed at 3479 (-)/3369 (+) cm-1,
and a mutation study revealed the bands to have originatedSuzuki et al.

FIGURE 9: SrSRIK minus SrSRI (a) and SrSRIM minus SrSRI (b)difference infrared spectra in the 3600-3000 cm-1 region, which
are multiplied by 1 and 0.66 from Figure 2a and Figure 6a,
respectively, to exhibit the same negative intensities at 1250 and
1200 cm-1. Underlined value corresponds to amide-A vibration.
One division of y axis corresponds to 0.0015 absorbance units.

from an O-H stretch of Thr204 (26). Interestingly, importance of Thr204 for negative phototaxis was revealed after
the FTIR observation (20). Thus, the high-frequency region
contains rich structural information related to function. In
the SrSRIK minus SrSRI spectrum, the bands at 3492 (-),
3392 (-), and 3357 (+) cm-1 would originate from O-H
stretching vibrations of threonine, tyrosine, or tryptophan
residue(s).
The N-H stretching vibrations of the peptide backbone(amide-A) also appear in this frequency region. Since N-H
and CdO groups form hydrogen bonds in the R-helix, we
expect a similar spectral feature for the amide-A and amide-I
regions, respectively, and this is indeed the case for the
SrSRIM minus SrSRI spectrum. Figure 9b shows peaks at3359 (+), 3334 (+), 3310 (-), 3278 (-), and 3255 (+) cm-1
in the amide-A region, while Figure 7a shows peaks at 1666(+), 1654 (-), 1614 (-), and 1605 (+) cm-1 in the amide-I
region. Thus, negative bands at 3310 and 1654 cm-1
correspond with each other in the R-helix, and the Mformation accompanies distortion of the R-helix as shownby the upshift to 3359 or 3334 cm-1 and 1666 cm-1,
respectively. On the other hand, negative bands at 3278 and1614 cm-1 also correspond with each other, and the Mformation leads to the spectral downshift. Unlike the former
case, the secondary structure is unclear from the frequencies,
where SrSRI may form a β-sheet, or some unusual structure.
DISCUSSION
Many organisms utilize light as a signal. SRI is one of
the most interesting photosensory receptors because of its
ability to mediate opposite signals depending on the color

FTIR spectroscopy of SrSRI
of light by use of photochromic reactions. However, themolecular mechanism for its function is not well understoodat the atomic level. Recently we successfully prepared SRI
from a eubacteria source (Salinibacter ruber) for the firsttime (to date found only in the haloarchaea). Most importantly, the SrSRI protein is stable even in the absence of
NaCl and amenable to study, unlike SRI from other sources(10). Here, we utilized FTIR spectroscopy to explore
structural changes of SrSRI and focused on similarities anddissimilarities between SrSRI and other rhodopsins todetermine what are important structural changes for functionof SRI.
The archaeon Halobacterium salinarum, a halophilicprokaryote, contains four retinal proteins, BR, HR, SRI, andSRII. The high-resolution crystal structures of BR, HR, andSRII and the SRII-HtrII complex were reported from 1997
through 2006 (5, 15, 52, 53), whereas the structure of SRI
has not been determined yet. While high stability of SrSRI(10) could lead to crystallization in the future, we will discuss
similarities and dissimilarities between SrSRI and otherrhodopsins based on the present FTIR observations.
Similarities and Dissimilarities in the Initial State. Proteinfunctions are entirely different among BR (proton pump),
HR (chloride pump), and SRII (negative phototaxis sensor),
but their X-ray crystallographic structures are very similar
among them (5, 15, 52, 53). Thus, it is reasonable to inferthat SRI has a similar protein architecture to other archaealtype rhodopsins. The present FTIR study indeed provides
this aspect, though FTIR spectroscopy cannot determine thetertiary structure. Similar retinal bands such as CdC and
C-C stretching vibrations in SrSRI and HsSRI (Figure 2)
to other rhodopsins imply a similar chromophore structure
in SRI. Negative amide-I bands at 1654 and 1647 cm-1 for
SrSRI and HsSRI (Figure 7), respectively, are characteristicof the R-helix, and absolute IR spectrum of SrSRI has a peakat 1657 cm-1 (data not shown). Thus, SRI probably possesses
an all-trans retinal chromophore in seven transmembrane
R-helices as do BR, HR, and SRII.
A correlation between proton pumping activity and existence of strongly hydrogen-bonded water molecule(s) (<2400
cm-1 as water O-D stretch) was discovered by Kandori’s
group by using various rhodopsins and their mutants (48).
Figure 5b,c shows that at <2400 cm-1, there are two waterbands for SRII (2307 and 2215 cm-1) and three water bands
for BR (2323, 2292, and 2171 cm-1), both of which pumpprotons. In contrast, strongly hydrogen-bonded water molecules were not observed for HR (Figure 5d), Neurospora
rhodopsin, and bovine rhodopsin (17, 36, 54, 55), which do
not pump protons. Figure 5a shows the presence of a water
O-D stretch at 2172 cm-1 for SrSRI, suggesting protonpump activity for SrSRI. In fact, light-induced pH changesof the SrSRI-containing vesicles revealed that SrSRI could
pump protons as well as BR and SRII (unpublished data).
A similar spectrum of SrSRI in the X-D stretchingvibrational region to those of BR and SRII (Figure 5),
namely, strongly hydrogen-bonded water O-D and Schiffbase N-D stretches, also suggest that a water molecule
bridges between the Schiff base and Asp72 in SrSRI.
Similarities and Dissimilarities in the K Intermediate.
Then, we focused on similarities and dissimilarities in the
K intermediate. The SrSRIK minus SrSRI spectrum is almost
identical to that of HsSRI (Figures 2-4), implying a similar

Biochemistry, Vol. 47, No. 48, 2008 12757
structure and structural changes upon formation of the K
intermediate between them. It appears that HOOP vibrations
are different between photosensors and ion pumps (Figure4), where HOOP vibrations of SRI and SRII are extended
widely along the retinal chromophore. In contrast, the HOOP
modes are more localized at the Schiff base region for BR
and HR. Spectral analysis of water bands shows thathydrogen-bonding destabilization is important for energy
storage in proton pumps, but energy storage for photosensory
receptors probably requires a more steric effect, such as asteric constraint between the C14-D group of retinal andThr204 in SRII (28).
In 2008, Ito et al. demonstrated a positive correlation
between vibrational amplitudes of the C14-H HOOP vibration at 864 cm-1 at 77 K (Figure 4c) and the physiologicalphototaxis response (28). The band was also observable at
861 cm-1 for BR-T (Figure 4d), but not for SrSRI (Figure4a). This suggests that the changes of C14-H HOOP are a
specific feature among sensors for negative phototaxis. It isreasonable because the counterpart of the C14 atom, Thr204
of SRII, is not conserved in other rhodopsins, including SRIs.
The distortion extended along the polyene chain of the retinal
in SRIs may be due to the photoisomerization-induced sterictrigger involving the retinylidene C13 methyl group previously demonstrated in HsSRI (56), even though the trigger
is a different one than in SRII.
The SrSRIK minus SrSRI spectrum at 3600-3000 cm-1
in D2O (Figure 9a) provides undeuterated O-H or N-H
vibrations. Since 2003, Kandori’s group have demonstrated
that the 110 cm-1 upshift of the O-H stretching vibrationof Thr204 in SRII and its alteration of hydrogen bond with
Tyr174 are required and essential for light-signal transduction
to HtrII (20, 26). In the case of BR, O-H stretchingvibrations of threonine (57) and N-H stretching vibrationsof tryptophan (58) were also studied in detail. Thus, it will
be important to identify the bands at 3492, 3392, and 3357
cm-1 in Figure 9a, which is one of our future focuses.
Similarities and Dissimilarities in the M Intermediate.
Because the active (signaling) intermediate of SRII-HtrIIand SRI-HtrI complexes is referred to as the M intermediate(2), it is important to analyze the structure and structuralchanges upon formation of the M intermediate. In the M
intermediate, appearance of a protonation signal of thecounterion is a common feature among rhodopsins (SRIs,
SRII, BR) except for HR, which has no M intermediate (59).
In SrSRI, the 1753 cm-1 band in H2O (1742 cm-1 in D2O)
is ascribable to a CdO stretching vibration of carboxylate(Figure 7a), and interpreted in terms of protonation of Asp72
by the proton transfer from the protonated Schiff base(Lys201) (Figure 10).
In addition, SrSRI has a negative band at 1743 cm-1 in
H2O (1734 cm-1 in D2O) (Figure 7a). A corresponding
positive peak is observed at 1414 (or 1397) cm-1 as a bandfor deprotonated carboxylate (Figure 6a). Therefore, we inferthat in SrSRI, a proton is released from the carboxylate to
the extracellular side, although a proton was released froma protonated water cluster in BR (22, 23).
The most different aspect between SRI and SRII is the
structural changes of the peptide backbone upon formationof the M intermediate (Figures 7 and 9). As described above,
the analysis of the M intermediate is important not only forphotosensors but also for ion-pumping rhodopsins, because

12758Biochemistry, Vol. 47, No. 48, 2008

FIGURE 10: (a) Schematic of the photochemical reaction cycle of
SrSRI identified in a previous study (10). Kt is the decay rate
constant of SrSRIM. (b) Structural changes upon formation of K
(purple) and M intermediate (yellow). Our results suggest that astrongly hydrogen-bonded water molecule exists between Asp72
and Lys201 in SrSRI. Upon formation of the K intermediate, retinal
isomerizes from the all-trans to the 13-cis form, and Asp102 isaffected by the structural changes. The primary proton transfer takes
place upon formation of the M intermediate. Then a proton istransferred from the protonated Schiff base (Lys201) to Asp72, anda proton may be released from a carboxylic residue.

of its role on the function. Frequency changes of amide-I
vibration of the R-helix were observed at 1666 (+)/1654 (-)
cm-1 in SrSRI, at 1661 (+)/1647 (-) cm-1 in HsSRI, andat 1663 (-)/1644 (+) cm-1 in SRII (Figure 7). Namely, thesebands in SRIs and in SRII showed a higher and lower
frequency shift, respectively. The amide-A vibrations at 3359(+) and 3334 (+)/3310 (-) cm-1 are likely to correspondto the amide-I vibrations at 1666 (+)/1654 (-) cm-1, whichprobe structural changes in the R-helix. The weakenedhydrogen bond of the peptide backbone in SRI is in contrast
to those in SRII. The different modes in protein structuralchanges between SRI and SRII may be correlated with theirfunction, dual receptors both for positive and negative
phototaxis versus a receptor for negative phototaxis.
ACKNOWLEDGMENT
We thank Dr. Mikihiro Shibata for providing HRK minus
HR infrared spectra and stimulus discussion, Dr. Jun Sasaki
for teaching us the method for preparing film samples of
HsSRI suitable for FTIR spectroscopy, and Dr. Tomomi
Kitajima-Ihara for encouragement.
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