6208

Biochemistry 2008, 47, 6208–6215

Steric Constraint in the Primary Photoproduct of Sensory Rhodopsin II Is a
Prerequisite for Light-Signal Transfer to HtrII†
Motohiro Ito,‡ Yuki Sudo,§,| Yuji Furutani,‡ Takashi Okitsu,⊥ Akimori Wada,⊥ Michio Homma,| John L. Spudich,§and Hideki Kandori*,‡
Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan, Center
for Membrane Biology, Department of Biochemistry and Molecular Biology, UniVersity of Texas Medical School, Houston,
Texas 77030, DiVision of Biological Science, Graduate School of Science, Nagoya UniVersity, Chikusa-ku, Nagoya 464-8602,
Japan, and Graduate School of Organic Chemistry for Life Science, Kobe Pharmaceutical UniVersity, Higashinada-ku,
Kobe 658-8558, Japan
ReceiVed February 29, 2008; ReVised Manuscript ReceiVed April 8, 2008

ABSTRACT: Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible fornegative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore fromall-trans to 13-cis initiates conformational changes in the protein, leading to activation of the cognate
transducer protein (HtrII). We previously observed enhancement of the C14-D stretching vibration of theretinal chromophore at 2244 cm-1 upon formation of the K state and interpreted that a steric constraint
occurs at the C14D group in SRIIK. Here, we identify the counterpart of the C14D group as Thr204, because
the C14-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C14-HOOP(hydrogen out-of-plane) vibration at 864 cm-1. Although the K state of the wild-type bacteriorhodopsin(BR), a light-driven proton pump, possesses neither 2244 nor 864 cm-1 bands, both signals appeared for
the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We founda positive correlation between these vibrational amplitudes of the C14 atom at 77 K and the physiologicalphototaxis response. These observations strongly suggest that the steric constraint between the C14 groupof retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.

Microbial rhodopsins convert light into chemiosmoticenergy or cellular signals (1, 2). The primary reaction is
all-trans to 13-cis photoisomerization of the retinal chromophore, and light energy is stored through altered
chromophore-protein interaction (2, 3). Such energy issubsequently consumed by changing the protein structures,and ion pumping or transducer activation is achieved in lightenergy or signal conversions, respectively. It is well-known
that retinal isomerization in the protein is a selective andefficient reaction along the potential surface of the excitedstate in rhodopsins (2, 3). One of the characteristics of thephotoisomerization is that the reaction occurs even at low†
This work was supported by grants from the Japanese Ministry of
Education, Culture, Sports, Science, and Technology to H.K. (19370067,
20044012, and 20050015) and Y.F. (19045015) and National Institutesof Health Grant R37GM27750 and a Robert A. Welch Foundation grant
to J.L.S.
* To whom correspondence should be addressed. Phone and Fax:
81-52-735-5207. E-mail: kandori@nitech.ac.jp.
‡
Nagoya Institute of Technology.
§
University of Texas Medical School.
|
Nagoya University.
⊥
Kobe Pharmaceutical University.
1
Abbreviations: SRII, sensory rhodopsin II from Natronomonas
pharaonis (also known as NpSRII or ppR); HtrII, halobacterialtransducer protein II; BR, bacteriorhodopsin; BR-T, triple mutant of
BR (P200T/V210Y/A215T) having photosensory signaling function;
SRIIK, K intermediate of SRII; BRK, K intermediate of BR; FTIR,
Fourier transform infrared; HOOP, hydrogen out-of-plane; PC, L-Rphosphatidylcholine; QM/MM, quantum mechanical/molecular mechanical.

temperatures. Since the protein motion is restricted at low
temperatures, the structure of the retinal binding site is hardly
altered. X-ray crystallography indeed showed little change
in the retinal pocket in the primary K intermediate ofbacteriorhodopsin (BR),1 a light-driven proton pump (4–6),
and pharaonis sensory rhodopsin II (SRII, also called
phoborhodopsin) (7), a light sensor for negative phototaxis,
in measurements at 100 K. Prior to protein structural changes,
the isomerized chromophore therefore has a distorted structure, by which light energy is stored.
How are such structural constraints observed experimentally? Current X-ray crystallographic structures of photointermediates are not sufficiently resolved for constructing thedetailed structure of the retinal chromophore. In fact, theretinal structure is considerably different among three X-ray
structures for the K intermediate of BR (4–6). The distorted
retinal chromophore has been best investigated throughvibrational analysis particularly on the hydrogen out-of-plane(HOOP) wagging vibrations in the 1000–800 cm-1 region(8). Previous resonance Raman spectroscopy of rhodopsins
successfully detected enhanced HOOP vibrations in theirprimary intermediates, and they were interpreted in termsof the origin of the chromophore distortion (8). Similar
enhanced HOOP modes were also observed for infrared
bands. Thus, it is generally accepted that HOOP vibrations
are a good probe of chromophore distortion in rhodopsins.
Recently, we reported a novel technique for monitoringsteric constraint of the retinal chromophore in the binding

10.1021/bi8003507 CCC: $40.75  2008 American Chemical Society
Published on Web 05/15/2008

Role of Steric Constraint between the C14 Group and Thr204

Biochemistry, Vol. 47, No. 23, 2008 6209
and Spudich successfully engineered BR into an SRII-likelight sensor by only three residue replacements, threonine
at SRII position 204 (Ala215 in BR) being the most critical(13). FTIR measurements of the triple mutant BR (BR-T)
revealed a frequency shift only in the complex with thetransducer protein, which can be ascribed to an O-H stretchof Thr215 which is the case for Thr204 of SRII (14). Thus,a crucial role of Thr204 is evident for light-signal transduction in SRII. Here we report that the C14D group indeed
interacts with Thr204 in SRIIK by use of several mutant
proteins. Such interaction was newly observed for the K stateof the light-sensing BR-T, but not for the wild-type BR.
Positive correlation between the vibrational amplitudes in
the K state and phototaxis responses strongly suggests thatthe steric constraint between the C14 group of retinal andThr204 of the protein is crucial for the activation of thetransducer protein.
MATERIALS AND METHODSFIGURE 1: (a) Chromophore molecule of SRII and BR. All-transretinal is bound to a lysine residue via a protonated Schiff baselinkage. In this study, the C14 atom is deuterated. (b) X-ray
crystallographic structure of SRII (PDB entry 1JGJ) in the extracellular-to-cytoplasmic view. Highlighted are the retinal chromophore (yellow) and residues that are mutated in this study. The
distance from the C14 atom to the phenol oxygen of Tyr174 is 4.0
Å, to the hydroxyl oxygen of Thr204 is 4.4 Å, to the hydroxyl
oxygen of Thr79 is 4.3 Å, and to the side chain nitrogen of Asn105
is 8.7 Å (7, 12). The OH group of Thr204 is within hydrogenbonding distance (3.2 Å) of the OH group of Tyr174.

pocket (9). In that study, all seven monodeuterated all-transretinal analogues (positions 7, 8, 10-12, 14, and 15) weresynthesized and incorporated into SRII (Figure 1a). The FTIR
measurements at 77 K revealed that only the C14-D
stretching vibration at 2244 cm-1 is significantly enhanced
upon formation of the K state. We interpreted that the stericconstraint occurs at the C14D group in SRIIK, where rotationof the C13dC14 bond probably leads to the movement of the
C14D group (9). It is noted that SRIIK exhibits various HOOP
vibrations, suggesting that the chromophore is widely
distorted along the polyene chain (10). In contrast to the
HOOP bands, the C-D stretching vibration was specific for
the C14 group of SRIIK (9). This fact implies that the C-D
stretch provides structural information about the directcontact of the C14 deuterium (hydrogen) with surroundings.
The counterpart of the C14D group in SRIIK is of interest.
According to the X-ray structure of SRII (11), the distancefrom the C14 atom to the phenol oxygen of Tyr174 is 4.0 Å,
to the hydroxyl oxygen of Thr204 is 4.4 Å, and to thehydroxyl oxygen of Thr79 is 4.3 Å (Figure 1b). The structureof SRIIK shows that only the distance to Thr204 is significantly reduced, from 4.4 to 3.3 Å, by retinal photoisomerization (7, 11). This result suggested that Thr204 may be thecounterpart of the C14D group (9). Interestingly, the functional importance of Thr204 has been emphasized by threearticles after the report in 2005. Phototaxis function was lostin the mutants of Thr204 or Tyr174, suggesting the biologicalsignificance of the hydrogen bond between them (12). Sudo

Preparation of the SRII Samples. The wild-type and mutant
proteins of SRII from Natronomonas pharaonis were prepared as described previously (15–17). Briefly, the SRII
protein with a histidine tag at the C-terminus was expressedin Escherichia coli, solubilized with 1.5% n-dodecyl β-Dmaltoside, and purified by Ni column chromatography. The
SRII samples with retinal deuterated at C14 (Figure 1) were
produced by adding all-trans-retinal (1 µM) labeled at C14
position to the E. coli culture instead of unlabeled all-transretinal (12). The purified SRII sample was then reconstituted
into L-R-phosphatidylcholine (PC) liposomes by the removalof the detergent with Biobeads, where the molar ratio of theadded PC to SRII was 50:1. The samples of SRII in PC
liposomes were finally washed twice [2 mM phosphate buffer(pH 7.0)] for the FTIR measurements.
Preparation of the BR Samples. The wild-type and triple
mutant [P200T/V210Y/A215T (BR-T)] proteins of BR wereexpressed in Halobacterium salinarum Pho81Wr- cells as
described previously (13). The purified purple membranesuspension was used for the FTIR measurements of theunlabeled retinal (18). On the other hand, the C14D-labeledretinal was incorporated as follows. Two molar hydroxylamine (pH 7.0) was added to the purple membrane pellet.
The sample was kept at room temperature and light througha cutoff filter (Y-52, >500 nm) focused onto the stirred
sample. The suspension became colorless after 1.5 h, andthe membrane was centrifuged and washed with 50 mM
phosphate buffer (pH 7.0, 200 mM NaCl) five times toremove any residual traces of hydroxylamine that could
interfere with the reconstitution reaction. The membrane was
finally resuspended with 50 mM phosphate buffer (pH 7.0,
200 mM NaCl). C14D-labeled retinal in an ethanolic solution
was added stepwise to the membrane suspension at room
temperature. The reconstitution efficiency was >75% forboth BR and BR-T. The BR and BR-T samples were finallywashed twice [2 mM phosphate buffer (pH 7.0)] for the FTIR
measurements.
FTIR Spectroscopy. FTIR spectroscopy was performed as
described previously (19, 20). Ninety microliters of the SRII
sample was dried on a BaF2 window with a diameter of 18
mm. The dry film was then rehydrated by putting an ∼1 µL
drop of water beside the film and sealing with another6210

Biochemistry, Vol. 47, No. 23, 2008

window and a silicon rubber O-ring (19, 20). After hydration,
the sample was placed in a cell, which was mounted in an
Oxford DN-1704 cryostat placed in the Bio-Rad FTS-40
spectrometer. All spectra were measured with 2 cm-1
resolution. The SRIIK minus SRII difference spectra were
measured as follows (20). Illumination of the SRII film at
pH 7 with 450 nm light at 77 K for 2 min converted SRII to
SRIIK, and subsequent illumination with >560 nm lightreverted SRIIK to SRII. On the other hand, BR and BR-T
were illuminated at 500 nm (2 min) and >640 nm (60 s) forformation and reversion of the K state, respectively (14).
The difference spectrum was calculated from spectra constructed with 128 interferograms collected before and after
the illumination, and sixteen spectra obtained in this way
were averaged for the difference spectra except for that of
Y174F. In the case of Y174F, the spectrum for photoreversion of SRIIK to SRII was not a mirror image of the SRIIK
minus SRII spectrum (i.e., the forward photoreaction). Since
Tyr174 is located next to the retinal chromophore, the Y-to-F
mutation must modify the local environment, leading to an
unusual photoconvesion. While the detailed mechanism
remains unclear, eight spectra independently obtained byillumination with 450 nm light were averaged to produce
the SRIIK minus SRII spectrum of Y174F in this study.
Phototaxis Analysis. Phototaxis responses were measured
as swimming reversal frequency changes of cell populations
in response to photostimulus as described previously (12, 21).
Swimming reversals were measured by stimulus-induced
effects on the ratio of rate of change of direction (RCD) to
speed (SPD) by computer-assisted motion analysis ofindividual cell tracks. The software used for the analysis
system was Celltrack 1.2 Beta (Motion Analysis Corp., Santa
Clara, CA); 200-2500 cells were assayed for each trace(Figure 4). Maximal stimuli from a 100 W Hg lamp througha heat filter and 40 nm band-pass interference filters delivered
through microscope optics were as follows: 100 ms, 500 nm
stimulus for wild-type SRII and SRII mutants; 100 ms, 580
nm stimulus for wild-type BR; and 100 ms, 550 nm stimulus
for BR-T (13). The HtrII transducer protein that forms acomplex with rhodopsin is from N. pharaonis in this study
except for the measurements of BR and BR-T, where H.
salinarum HtrII was used (13). The expression levels
calculated from amplitudes of the pigments’ main absorption
bands in the visible spectrum for SRII-HtrII, BR-T-HtrII,
T204A-HtrII, T204S-HtrII, T204C-HtrII, Y174F-HtrII,
T79A-HtrII, N105D-HtrII, and BR-HtrII complexes intransformants used in this study were all similar, namely,
2.0 × 104, 1.0 × 104, 2.7 × 104, 1.8 × 104, 1.7 × 104, 1.1
× 104, 1.8 × 104, 2.1 × 104, and 3.5 × 104 molecules percell, respectively.
RESULTSC-D Stretching Vibrations of the Retinal in SRIIK of
Mutants Show the Steric Constraint of the C14 Atom andThr204. Figure 2A shows the SRIIK minus SRII difference
spectra of the wild type (a) and several mutants (b-g) in
the C-D stretching region. As reported previously (9), thespectra of wild-type SRII exhibit a strong positive peak at
2244 cm-1 for the C14D-labeled retinal (solid line in part aof Figure 2A), but not for the unlabeled retinal (dotted line
in part a of Figure 2A). Such a C-D stretch was unique for

Ito et al.

FIGURE 2: (A) SRIIK minus SRII difference spectra of the wild type(a), T204A (b), T204S (c), T204C (d), Y174F (e), T79A (f), andN105D (g) in the 2330-2190 cm-1 region. The solid line represents
the spectrum for the C14D-labeled retinal, while the dotted line
corresponds to that for the unlabeled retinal. Vertical amplitudesof all spectra are normalized by the bands of Asn105 at 1704 (-)/
1700 (+) cm-1 for spectra a-f. On the other hand, those in spectrum
g were normalized to the wild type (a) by the C-C stretch at 1204(-) cm-1 (unlabeled retinal) or the bands at 1743 (-)/1738 (+)
cm-1 (C14D-labeled retinal) (see Figure S1 in detail). One divisionof the y-axis corresponds to 0.00018 absorbance unit. (B) SRIIK
minus sRII difference spectra of the wild type (a), T204A (b),T204S (c), T204C (d), Y174F (e), T79A (f), and N105D (g) in the880-845 cm-1 region. The solid line represents the spectrum forthe unlabeled retinal, while the dotted line corresponds to that for
the C14D-labeled retinal. One division of the y-axis corresponds to
0.0008 absorbance unit.

the C14D group, and no peaks were observed for the C7D,
C8D, C10D, C11D, C12D, and C15D groups (9). The lack of acorresponding negative peak suggests that the C14-D stretch
for SRII is much smaller in amplitude than in SRIIK. Thus,
it is likely that only the C14-D stretching vibration is greatly
enhanced upon retinal photoisomerization of SRII, and weinterpreted that the enhanced C–D stretch originates fromthe steric constraint with the surrounding protein moiety.
Then, we tested several mutant proteins. The SRIIK minus
SRII difference spectra in the 1800-800 cm-1 region showthat retinal photoisomerization takes place in all mutants asin the wild type (Figure S1 of the Supporting Information).
Spectra b-d of Figure 2A show that the positive band
completely disappears for the mutant of Thr204, implyingthat the counterpart of the steric interaction of the C14D groupis Thr204. Spectrum e of Figure 2A shows that the positive
band is significantly reduced in intensity also in the Y174F
mutant. Since Tyr174 forms a hydrogen bond with Thr204
in SRII, the lack of a hydrogen bond in the Y174F mutant
may influence the steric interaction between the C14 groupand Thr204. In other words, these results strongly suggestthat the counterpart of the C14D group is Thr204, not Tyr174.
This is consistent with the X-ray structures of SRII andSRIIK, where the distance from the C14 atom changes fromonly 4.0 to 3.9 Å for the phenol oxygen of Tyr174, and alarger change from 4.4 Å to 3.3 Å occurs for the hydroxyl
oxygen of Thr204 (7, 11). It also should be noted that thepeak frequency is upshifted to 2251 cm-1 for Y174F, andRole of Steric Constraint between the C14 Group and Thr204
the broadened band implies conformational heterogeneity for
the C14D group (spectrum e of Figure 2A). The positive peak
was preserved but considerably reduced in magnitude for
T79A (spectrum f of Figure 2A), whose peak frequency wasslightly upshifted (2246 cm-1). In contrast, the positive band
looks identical for N105D (spectrum g of Figure 2A),
although the peak intensity was slightly reduced.
Hydrogen Out-Of-Plane (HOOP) Vibrations of the Retinal
in SRIIK of Mutants Show the Steric Constraint of the C14
Atom and Thr204. HOOP vibrations are also regarded as
the probe of the distortion of the retinal chromophore (8).
Therefore, if the steric constraint occurs at the C14 group,
the C14-HOOP mode should also be influenced. We previously identified the positive band at 864 cm-1 as the
C14-HOOP mode, because the 864 cm-1 band disappeared
only in the C14D spectrum (10). Spectrum a of Figure 2B
reproduces the previous result, where the positive band at
864 cm-1 (solid line) significantly reduces the intensity for
the C14-D labeling (dotted line). The presence of the
C14-HOOP band for SRIIK can be interpreted in terms ofthe chromophore distortion at this moiety. The positive peakat 864 cm-1 significantly reduces the intensity for T204A,
T204S, T204C, and Y174F (spectra b-e of Figure 2B) whichis the case for the C14-D stretching vibrations (spectra b-eof Figure 2A). The 864 cm-1 band was preserved in T79A,
but the intensity became approximately half (spectrum f of
Figure 2B). The positive band looks identical for N105D(spectrum g of Figure 2B), although the peak intensity wasslightly reduced. These spectral features for T79A and
N105D were also the same as for the C14-D stretchingvibrations (spectra f and g of Figure 2A).
Since many HOOP and other vibrations exist in thisfrequency region, the spectra are not as clear as those for
the C-D stretching vibrations. However, the spectral featureof the C14-HOOP band at 864 cm-1 is consistent with thatof the C14-D stretching band at 2244 cm-1. It should be
noted that the C14-HOOP mode is located at a frequency
33 cm-1 higher than that of the 13-cis-retinal (831 cm-1)(22) and is closer to that of the all-trans-retinal (876 cm-1)(23). This indicates that the chromophore of SRIIK is in an
unrelaxed 13-cis configuration.
C14-D Stretching and C14-HOOP Vibrations in BR-TK.
BR-T activates HtrII, and the BR mutant gains light signaling
activity (13). How do the vibrations related to the C14 atom
in BR-T differ from those of wild-type BR? In this study,
we incorporated C14D-labeled retinal into wild-type BR and
BR-T, and the isotope effect was examined by measuring
the difference FTIR spectra between the K and unphotolyzed
states. Unlike SRII, the wild-type BR containing C14Dlabeled retinal exhibits no positive bands in the frequency
region of C-D stretching (solid line in part a of Figure 3A),
indicating that the 2244-cm-1 band is unique for SRII. In
other words, formation of the K state does not entail stericconstraint around the C14 atom in wild-type BR. In contrast,
BR-T containing the C14-D labeled retinal shows an appearance of the band at 2250 cm-1 (solid line in part b of Figure
3A), which is absent for the unlabeled one (dotted line inpart b of Figure 3A). The corresponding amino acid toThr204 in SRII is Ala215 in BR, and Ala215 is replacedwith threonine in BR-T (P200T/V210Y/A215T). Thus, thenewly appearing C14-D stretch in part b of Figure 3A
apparently originates from the steric constraint between the

Biochemistry, Vol. 47, No. 23, 2008 6211

FIGURE 3: (A) Difference spectra of BR (a) and BR-T (b) in the2300-2210 cm-1 region. The solid line represents the spectrum
for the C14D-labeled retinal, while the dotted line corresponds tothat for the unlabeled retinal. Vertical amplitudes are normalized
by the bands to wild-type SRII (Figure 2a) by the C-C stretch at1204 (-) cm-1 (unlabeled retinal). Those for the C14D-labeledretinal are then normalized to those for the unlabeled retinal by
the amide I bands at 1639 (-)/1623 (+) (a) and 1640 (-)/1623(+) (b) cm-1 (see Figure S1 in detail). One division of the y-axis
corresponds to 0.0003 absorbance unit. (B) Difference spectra of
BR (a) and BR-T (b) in the 900-780 cm-1 region. The solid line
represents the spectrum for the unlabeled retinal, while the dottedline corresponds to that for the C14D-labeled retinal. One divisionof the y-axis corresponds to 0.002 absorbance unit.

C14 atom and Thr215 in BR-T. It should be noted that we
did not examine the single A215T mutant in this study.
However, the spectrum of the K intermediate in the P200T/
V210Y mutant BR is almost identical to that of wild-type
BR (14), implying that the observed C14-D stretch arises
from direct interaction with Thr215.
Parts a and b of Figure 3B show the HOOP region of wildtype BR and BR-T, respectively. We already reported thespectra for the unlabeled retinal (solid lines in parts a and b
of Figure 3B) (14), while the spectra for the C14D-labeledretinal are newly measured (dotted lines). Two peaks at 811
and 803 cm-1 in part a of Figure 3B almost disappear upondeuteration of C14. Therefore, wild-type BR possesses the
C14-HOOP mode at these frequencies, which is consistent
with the previous resonance Raman study (24). In contrast,
BR-T does not exhibit the bands at 811 and 803 cm-1.
Instead, a new band appeared at 861 cm-1 (part b of Figure3B) that is close in frequency to the C14-HOOP mode ofwild-type SRII (part a of Figure 2B). In fact, the bandsignificantly reduces the intensity for the C14-D labeling(dotted line), indicating that the band contains the C14HOOP vibration.
Thus, both C-D stretching and HOOP vibrations in BR-T
are fully consistent with the observations for SRII, indicatingthat the K state of BR-T exhibits a steric constraint at theC14 atom of retinal with Thr215. Therefore, BR-T wasconverted from the BR-type difference spectrum to the SRIItype difference spectrum by introduction of this stericconstraint with the retinal chromophore (13). It should be
noted that wild-type BR possesses the C14-HOOP mode at
811 and 803 cm-1 (part a of Figure 3B), but no C-D
stretching vibrations (part a of Figure 3A), indicating that

6212

Biochemistry, Vol. 47, No. 23, 2008

FIGURE 4: Phototaxis responses measured as swimming reversal
frequency changes to photostimulus (arrow): 100 ms, 500 nm
stimulus for wild-type SRII and SRII mutants and 100 ms, 550 nm
stimulus for wild-type BR and BR-T. The swimming reversal
frequency in a cell population is monitored by the quotient of therate of change of direction (RCD) and linear swimming speed(SPD), which increase and decrease, respectively, during a swimming reversal. Cells contain the wild-type SRII-HtrII (a),T204A-HtrII (b), T204S-HtrII (c), T204C-HtrII (d), Y174F-HtrII(e), T79A-HtrII (f), and N105D-HtrII (g) complexes and the wildtype BR-HtrII (h) and BR-T-HtrII (i) complexes.

the C-D stretch is more specific to the structural change ofthe retinal chromophore.
Phototaxis Responses of the Cells Containing Mutant SRII
Receptors. FTIR spectra show how the mutations affect earlyphotocycle structural changes in SRII, BR, and BR-T. We
measured the influence of each mutation on phototaxis
responses by the motility behavior of cells containing the
mutant proteins. Figure 4a shows that the SRII-HtrII
complex mediated a phototaxis response evident as atransient increase in swimming reversal frequency. As wereportedpreviously,theT204A-HtrII(Figure4b),T204S-HtrII(Figure 4c), and Y174F-HtrII (Figure 4e) complexes did
not mediate detectable responses, while the T204C-HtrII(Figure 4d) complex mediated a phototaxis response similar
to that of the wild type (12). T79A and N105D mutants also
exhibited phototaxis responses (Figure 4f,g).
Parts h and i of Figure 4 show the results for BR and BRT, respectively. The former is a negative control, because
BR does not form a complex with HtrII. On the other hand,
BR-T forms a complex with HtrII and mediates negative
phototaxis. Phototaxis responses from BR-T were found to
be much greater with HtrII from H. salinarum than with HtrIIfrom N. pharaonis, possibly because of tighter or more

Ito et al.

FIGURE 5: Correlation between the C14-D stretching (a) or
C14-HOOP (b) signals in the K photointermediate and Km valuesof phototaxis responses. The Km values were calculated fromMichaelis-Menten fits to the fluence-response curves as describedin ref 14. (a) The abscissa corresponds to the normalized area ofthe positive bands at 2244 cm-1 in Figures 2A and 3A. The straightline is the best linear fit to the data excluding the T204 mutants
and BR-T. (b) The abscissa corresponds to the subtracted amplitudeof the C14D-labeled retinal HOOP (dotted line) from the unlabeledretinal HOOP (solid line) at 864 cm-1 in Figures 2B and 3B. The
straight line is the best linear fit to the data except for T204C and
BR-T. The values for the wild-type SRII are normalized to unity
in panels a and b. Dashed circles represent the corrected data forthe all-trans form of BR-T, where the phototaxis response isdoubled because BR-T possesses approximately half of the 13-cis
form.

efficient coupling (13), and Figure 4i therefore shows theresult with HtrII from H. salinarum.
Correlation between the Amplitudes of the Vibrations at
the C14 Atom and Phototaxis Responses. The C14-D stretching and C14-HOOP bands are observed for the K state,
which appears on the picosecond (10-12) time scale. On theother hand, phototaxis responses appear on a subsecond(10-1) time scale (Figure 4). In spite of such different timescales, vibrational and phototaxis signals seem to be correlated for the mutants tested. The fraction of light-induced
reversing of cells containing a mutant SRII-HtrII complex
used in this study was plotted against photostimulus duration(2-500 ms) (13), and the Km values from Michaelis-Menten
fits to the fluence-response curves were estimated assensitivities of cells expressing BR-T-HtrII, T204A-HtrII,
T204S-HtrII, T204C-HtrII, Y174F-HtrII, T79A-HtrII,
and N105D-HtrII complexes (35, 0, 0, 88, 0, 47, and 82%,
respectively, of that conferred by the wild-type SRII-HtrII
complex). Figure 5a plots the Km values versus their C14-D
stretching amplitudes, where the signal of the wild-type SRII
is normalized to unity. The x-axis of Figure 5a represents
the area of the difference spectra between C14D-labeled (solidRole of Steric Constraint between the C14 Group and Thr204
lines) and unlabeled (dotted lines) retinals in Figure 2A. The
straight line in Figure 5a implies a linear correlation between
the C14-D stretching amplitude and phototaxis response
except for the mutants of Thr204 and BR-T. Figure 5b
similarly plots the Km values of SRII and BR-T versus their
C14-HOOP signals, where the signal of the wild-type SRII
is normalized to unity. Unlike the C14-D stretching vibration,
deuteration of the C14 atom shows complex isotope effects,
because there are multiple vibrations containing the C14HOOP mode. Therefore, the x-axis of Figure 5b simply
represents the subtracted amplitude of the C14D-labeled
HOOP (dotted lines in Figures 2B and 3B) from unlabeled(solid lines) retinals at 864 cm-1. We observe a linear
dependence except for T204C and BR-T. It should be notedthat BR-T deviates from the straight line but BR-T possesses
approximately half of the 13-cis form in the dark. If the lowcontent of the all-trans form is corrected (dashed circles in
Figure 5), positive correlations are also obtained for BR-T.
These results strongly suggest that, if the steric constraint at
the C14 position is weakened, receptor phototaxis signals
become smaller.
Figure 5 also suggests some differences for the C14-D
stretching (a) and C14-HOOP (b) vibrations. The Y174F
mutant exhibits a small C14-D stretch (part e of Figure 2A),
but no C14-HOOP vibration (part e of Figure 2B). As aconsequence, the straight line in Figure 5a exhibits an offset
at y ) 0, while the straight line in Figure 5b approximately
crosses the origin. Such a difference may originate from thediffering structural information from the C-D stretch andthe HOOP vibration, though both monitor steric constraint.
As noted above, the counterpart of the C14D (H) group isprobably the side chain of Thr204, and it is likely that theC-D vibration monitors more local events. Therefore, themutants of Thr204 may have to be excluded from the linear
correlation in the C-D stretch (Figure 5a), whereas theresults for T204A and T204S are on the straight line for the
HOOP vibration (Figure 5b).
T204C does not follow the correlation in Figure 5. There
are neither C14-D stretching nor C14-HOOP vibrations for
T204C (Figure 2d), but this mutant mediates normal phototaxis responses (Figure 4d). Apparently, the light-signaltransduction in T204C does not need the steric constraint of
the C14 atom of retinal. One possibility is that a similar stericconstraint in T204C is already relaxed at 77 K, and lower
temperatures would be required to observe C14-D stretchingor C14-HOOP vibrations for T204C. Another possibility isthat T204C, but not T204A or T204S, mimics the structurenear position 204 found in the wild type after isomerizationof the retinal and after the steric constraint is resolved. In
other words, T204C may exhibit the local structure characteristic of the photoactivated state. However, the FTIR spectra
at 77 K are otherwise similar between the wild type and
T204C (Figure S1), suggesting the formation of the normal
K state for T204C. Therefore, the intramolecular signaltransduction mechanism of T204C is unclear at present.
In summary, the C14-D stretching and C14-HOOP bands
are positively correlated with the phototaxis response in thepresence of Thr204, the counterpart of the C14 group. Since
their time scales are widely separated (10-12 s vs 10-1 s),
we infer that the light-energy storage in the steric constraint
at the retinal C14 group is a prerequisite for protein structuralchanges for the activation of HtrII, leading eventually to the

Biochemistry, Vol. 47, No. 23, 2008 6213
function, phototaxis. Each mutation at Tyr174, Thr79, andAsn105 apparently weakens the chromophore-protein interaction, thereby reducing the phototaxis response. In otherwords, effects of mutation on the function may be explainedin terms of light-energy storage in the steric constraint.
DISCUSSION
C14-D Stretching and C14-HOOP Vibrations in the K
Intermediate. This study supports the notion that the enhanced C14-D stretching vibration at 2244 cm-1 in SRIIK
originates from the interaction between the C14D group of
the chromophore and Thr204 (Figure 2A). The enhanced
C-D stretch must result from an enlarged dipole momentof the vibration, and we identify the local steric constraint
between the C14D group and the side chain of Thr204 as theorigin of the strong electric polarization. It should be notedthat direct steric conflict may not occur, but rather a strong
interaction between the C14D group and Thr204 may directly
cause such electric polarization of the C14D group. While
the mechanism of the enlarged dipole must be examinedtheoretically, the positive correlation between the C14-D
stretching amplitude and phototaxis responses (Figure 5a)strongly suggests that the enhanced C14-D stretching vibration is functionally important.
Steric constraint between the C14D group of the chromophore and Thr204 is also supported by the analysis of
the C14-HOOP vibration at 864 cm-1. The C14-HOOP
signals similarly diminished in the T204 mutants as well as
Y174F (Figure 2B). We also observed a positive correlationof the C14-HOOP amplitude with the phototaxis response(Figure 5b). Thus, both C14-D stretch and C14-HOOP
vibrations imply that the chromophore-protein interaction
at the C14 position may be critical for protein function.
Despite the common information gained from the vibrational
analysis of the C14-D stretching and C14-HOOP vibrations,they are not necessarily identical. Previous study showed thatthe band at 864 cm-1 also contains a C12-HOOP vibration,
because the amplitude is considerably reduced for C12Dlabeled retinal (10). Thus, the band at 864 cm-1 originatesfrom the coupled HOOP vibrations of C14 and C12 atoms,
while the C14-HOOP vibration is predominant. On the other
hand, the C-D stretching vibration is specific to the C14
group (9), implying that the C-D stretch provides structural
information about the direct contact of deuterium (hydrogen)with surrounding atoms.
Mechanism of Light-Energy Storage and Signal Transduction. Our results argue that steric constraint at the retinal
C14 group is likely a prerequisite for protein structuralchanges for the activation of HtrII, leading eventually to thefunction, phototaxis. Figure 6 schematically illustrates apossible intramolecular and intermolecular signal relay
pathway based on the SRII-HtrII complex structure. In thismodel, retinal isomerization causes the steric conflict at theretinal C14 group with the side chain of Thr204, whichstrongly perturbs the hydrogen bonds of Thr204 with thephenol oxygen of Tyr174 and the peptide backbone ofLeu200 (Figure 6). Such structural perturbation is relayed
into the intermolecular hydrogen bond between Tyr199 of
SRII and Asn74 of HtrII and/or nearby van der Waals contact
regions (15, 25). The correlation in Figure 5 strongly suggests

6214

Biochemistry, Vol. 47, No. 23, 2008

FIGURE 6: Schematic drawing of the primary step in the light-signaltransduction in SRII (PDB entry 1H2S). Retinal photoisomerization
causes the steric constraint between the C14H group and Thr204,
by which the C14-D stretching vibration is enhanced. Light energy
stored by the steric constraint is used for changing the protein
structures, including the protein-protein interaction surface with
HtrII.

that the energy storage in the early intermediate is aprerequisite for the subsequent functional process.
With regard to the mechanism of light-energy storage,
recent findings on the proton-pumping rhodopsins should beconsidered. Low-temperature FTIR studies showed a strong
correlation between the presence of strongly hydrogenbonded water molecule(s) and the proton pumping activityof rhodopsins (26). For example, strongly hydrogen-bonded
water molecules are observed for BR (27), SRII (19),proteorhodopsin (28), Leptosphaeria rhodopsin (29), andazide-bound halorhodopsin (30), which all pump protons.
Strongly hydrogen-bonded water molecules were not observed for salinarum halorhodopsin (31), pharaonis halorhodopsin (32), Anabaena sensory rhodopsin (33), Neurospora rhodopsin (34), or bovine rhodopsin (35), which haveno proton pumping activity. Cl--pumping BR_D85S has nostrongly hydrogen-bonded water molecules (36), but protonpumping BR_D212N(Cl-) has strongly hydrogen-bonded
water molecules (37). These results were interpreted in termsof the proposal that the presence of a strong hydrogen bondof water is a prerequisite for proton pumping in rhodopsins(26). It is likely that destabilization of a water-containinghydrogen-bonding network plays an important role in lightenergy storage in this case.
It should be noted that SRII (19) and the SRII-HtrII
complex (38) both possess strongly hydrogen-bonded watermolecules, whereas the latter does not pump protons but
functions in light-signal transduction. Phylogenetic analysis
suggests that rhodopsin photosensors like SRI and SRII
evolved from light-driven proton pumps (39). Therefore,
sensory rhodopsins can be considered pumps that have gained
a mechanism for activating transducer proteins. This study
indicates that photoisomerization-induced steric constraint
between the C14H group of the retinal chromophore andThr204 in SRII is one such mechanism.
The light-energy storage capacity of microbial rhodopsinsis smaller than that in bovine rhodopsin (3). Low-temperature
photocalorimetric studies reported the energy stored in the
primary intermediates is 35 and 16 kcal/mol for bovine

Ito et al.
rhodopsin (40) and BR (41), respectively. This indicates that
only 30% of the light energy is stored in the structure of
BRK, which is approximately half of that in visual rhodopsin(60%). The loss of 70% of the light energy in the formationof BRK does not compromise BR function, since proton
pumping by BR is a quantal process; i.e., BR pumps a single
proton by use of one photon, and the free energy gain bypumping a proton is 6 kcal/mol (2, 3). The presence ofstrongly hydrogen-bonded water molecule in the protonpumping rhodopsins suggests that hydrogen bonding stabilization of a water molecule and its destabilization in the Kstate is crucial for the pumping function. In fact, previous
QM/MM calculations of BR and BRK estimated the hydrogenbonding destabilization to be 11 kcal/mol (70%) of the totalenergy storage of 16 kcal/mol (42, 43). On the other hand,
opto-acoustic spectroscopy revealed that the enthalpy change
upon formation of SRIIK (21 kcal/mol) is larger than thatfor BRK (13 kcal/mol in this study) (44, 45). Thus, it is likelythat the steric constraint at the C14 atom of retinal contributes
to additional energy storage, acquired during the evolutionof SRII from a BR-like pump, by the introduction of Thr204.
This inference is supported by the fact that BR-T activates
HtrII, demonstrating that BR and SRII share their fundamental mechanisms of phototransduction (13).
SUPPORTING INFORMATION AVAILABLESRIIK minus SRII difference FTIR spectra of the wild type,
T204A, T204S, T204C, Y174F, T79A, and N105D and BRK
minus BR difference FTIR spectra of the wild type and BR-T
in the 1790-810 cm-1 region, where the chromophore
molecule is the unlabeled and C14D-labeled retinal (FigureS1). This material is available free of charge via the Internet
at http://pubs.acs.org.
REFERENCES
1. Spudich, J. L., and Lanyi, J. K. (1996) Shuttling between two
protein conformations: The common mechanism for sensorytransduction and ion transport. Curr. Opin. Cell Biol. 8, 452–457.
2. Kandori, H. (2006) in Retinal Binding Proteins: From cis-transIsomerization in Biochemistry (Dugave, C., Ed.) Chapter 4, pp 53,
Wiley-VCH: Freiburg, Germany.
3. Birge, R. R. (1990) Nature of the primary photochemical eventsin rhodopsin and bacteriorhodopsin. Biochim. Biophys. Acta 1016,
293–327.
4. Edman, K., Nollert, P., Royant, A., Belrhali, H., Pebay-Peyroula,E., Hajdu, J., Neutze, R., and Landau, E. M. (1990) High-resolution
X-ray structure of an early intermediate in the bacteriorhodopsin
photocycle. Nature 401, 822–826.
5. Schobert, B., Cupp-Vickery, J., Hornak, V., Smith, S., and Lanyi,
J. (2002) Crystallographic Structure of the K Intermediate of
Bacteriorhodopsin: Conservation of Free Energy after Phtoisomerization of the Retinal. J. Mol. Biol. 321, 715–726.
6. Matsui, Y., Sakai, K., Murakami, M., Shiro, Y., Adachi, S.,
Okumura, H., and Kouyama, T. (2002) Specific Damage Inducedby X-ray Radiation and Structural Changes in the Primary
Photoreaction of Bacteriorhodopsin. J. Mol. Biol. 324, 469–481.
7. Edman, K., Royant, A., Nollert, P., Maxwell, C. A., PebayPeyroula, E., Navarro, J., Neutze, R., and Landau, E. M. (2002)
Early Structural Rearrangements in the Photocycle of an Integral
Membrane Sensory Receptor. Structure 10, 473–482.
8. Mathies, R. A., Smith, S. O., and Palings, I. (1987) in Biologicalapplications of Raman spectroscopy: Resonance Raman spectraof polyenes and aromatics (Spiro, T. G., Ed.) Vol. II, pp 59-108,
John Wiley and Sons, New York.
9. Sudo, Y., Furutani, Y., Wada, A., Ito, M., Kamo, N., and Kandori,
H. (2005) Steric Constraint in the Primary Photoproduct of an
Archaeal Rhodopsin from Regiospecific Perturbation of C-D

Role of Steric Constraint between the C14 Group and Thr204
Stretching Vibration of the Retinyl Chromophore. J. Am. Chem.
Soc. 127, 16036–16037.
10. Furutani, Y., Sudo, Y., Wada, A., Ito, M., Shimono, K., Kamo,
N., and Kandori, H. (2006) Assignment of the Hydrogen-Out-OfPlane and -in-Plane Vibrations of the Retinal Choromophore in
the K Intermediate of pharaonis Phoborhodopsin. Biochemistry 45,11836–11843.
11. Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., and Spudich,
J. L. (2001) Crystal Structure of Sensory Rhodopsin II at 2.4 Å:
Insights into Color Tuning and Transducer Interaction. Science 293,
1499–1503.
12. Sudo, Y., Furutani, Y., Kandori, H., and Spudich, J. L. (2006)
Functional Importance of the Interhelical Hydrogen Bond between
Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration during
the Signaling Process. J. Biol. Chem. 281, 34239–34245.
13. Sudo, Y., and Spudich, J. L. (2006) Three strategically placedhydrogen-bonding residues convert a proton pump into a sensory
receptor. Proc. Natl. Acad. Sci. U.S.A. 103, 16129–16134.
14. Sudo, Y., Furutani, Y., Spudich, J. L., and Kandori, H. (2007) Early
Photocycle Structural Changes in a Bacteriorhodopsin Mutant
Engineered to Transmit Photosensory Signals. J. Biol. Chem. 282,
15550–15558.
15. Sudo, Y., Furutani, Y., Shimono, K., Kamo, N., and Kandori, H.
(2003) Hydrogen Bonding Alteration of Thr-204 in the Complex
between pharaonis Phoborhodopsin and Its Transducer Protein.
Biochemistry 42, 14166–14172.
16. Iwamoto, M., Sudo, Y., Shimono, K., Araiso, T., and Kamo, N.
(2005) Correlation of the O-Intermediate Rate with the pKa of Asp75 in the Dark, the Counterion of the Schiff Base of Pharaonis
Phoborhodopsin (Sensory Rhodopsin). Biophys. J. 88, 1215–1223.
17. Kandori, H., Shimono, K., Shichida, Y., and Kamo, N. (2002)
Interaction of Asn 105 with the Retinal Chromophore during
Photoisomerization of pharaonis phoborhodopsin. Biochemistry 41,4554–4559.
18. Tanimoto, T., Furutani, Y., and Kandori, H. (2003) Strucral
Changes of Water in the Schiff Base Region of Bacteriorhodopsin:
Proposal of Hydration Switch Model. Biochemistry 42, 2300–2306.
19. Kandori, H., Furutani, Y., Shimono, K., Shichida, Y., and Kamo,
N. (2001) Internal Water Molecules of pharaonis Phoborhodopsin
Studied by Low-Temperature Infraed Spectroscopy. Biochemistry40, 15693–15698.
20. Kandori, H., Shimono, K., Sudo, Y., Iwamoto, M., Shichida, Y.,
and Kamo, N. (2001) Structural Changes of pharaonis Phoborhodopsin upon Photoisomerization of the Retinal Chomophore:
Infrared Spectral Comparison with Bacteriorhodopsin. Biochemistry40, 9238–9246.
21. Jung, K. H., and Spudich, J. L. (1996) Protonatable residues atthe cytoplasmic end of transmembrane helix-2 in the signaltransducer HtrI control photochemistry and function of sensory
rhodopsin I. Proc. Natl. Acad. Sci. U.S.A. 93, 6557–6561.
22. Curry, B., Palings, I., Broek, A., Pardoen, J. A., Mulder, P. P. J.,
Lugtenbrug, J., and Mathies, R. (1984) Vibrational Analysis of
13-cis-Retinal. J. Phys. Chem. 88, 688–702.
23. Curry, B., Broek, A., Lugtenbrug, J., and Mathies, R. (1982)
Vibrational Analysis of all-trans-Retinal. J. Am. Chem. Soc. 104,
5274–5286.
24. Palings, I., van den Berg, E. M., Lugtenburg, J., and Mathies, R. A.
(1989) Complete Assignment of the Hydrogen Out-of-Plane
Wagging Vibrations of Bathorhodopsin: Chromophore Structureand Energy Storage in the Primary Photoproduct of Vision.
Biochemistry 28, 1498–1507.
25. Furutani, Y., Kamada, K., Sudo, Y., Shimono, K., Kamo, N., andKandori, H. (2005) Structural Changes of the Complex between
pharaonis Phoborhodopsin and Its Cognate Transducer upon
Formation of the M Photointermediate. Biochemistry 44, 2909–
2915.
26. Furutani, Y., Shibata, M., and Kandori, H. (2005) Stronglyhydrogen-bonded water molecules in the Schiff base region ofrhodopsin. Photochem. Photobiol. Sci. 4, 661–666.

Biochemistry, Vol. 47, No. 23, 2008 6215
27. Shibata, M., Tanimoto, T., and Kandori, H. (2003) Water Moleculesin the Schiff Base Region of Bacteriorhodopisn. J. Am. Chem. Soc.
125, 13312–13313.
28. Furutani, Y., Ikeda, D., Shibata, M., and Kandori, H. (2006)
Strongly hydrogen-bonded water molecules is observed only inthe alkaline form of proteorhodopsin. Chem. Phys. 324, 705–708.
29. Sumii, M., Furutani, Y., Waschuk, S. A., Brown, L. S., andKandori, H. (2005) Strongly Hydrogen-Bonded Water Molecule
Present near the Retinal Chromophore of Leptosphaeria Rhodopsin,
the Bacteriorhodopsin-like Proton Pump from a Eukaryote. Biochemistry 44, 15159–15166.
30. Muneda, N., Shibata, M., Demura, M., and Kandori, H. (2006)
Internal Water Molecules of the Proton-pumping Halorhodopsin
in the Presence of Azide. J. Am. Chem. Soc. 128, 6294–6295.
31. Shibata, M., Muneda, N., Ihara, K., Sasaki, T., Demura, M., andKandori, H. (2004) Internal water molecules of light-driven choride
pump proteins. Chem. Phys. Lett. 392, 330–333.
32. Shibata, M., Muneda, N., Sasaki, T., Shimono, K., Kamo, N.,
Demura, M., and Kandori, H. (2005) Hydrogen-Bonding Alterations of the Protonated Schiff Base and Water Molecule in the
Chloride Pump of Natronobacterium pharaonis. Biochemistry 44,12279–12286.
33. Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005)
FTIR Spectroscopy of the All-Trans Form of Anabaena Sensory
Rhodopsin at 77 K: Hydrogen Bond of a Water between the Schiff
Base and Asp75. Biochemistry 44, 12287–12296.
34. Furutani, Y., Bezerra, A. G., Jr., Waschuk, S., Sumii, M., Brown,
L. S., and Kandori, H. (2004) FTIR Spectroscopy of the K
Photointermediate of Neurospora Rhodopsin: Structural Changesof the Retinal, Protein, and Water Molecules after Photoisomerization. Biochemistry 43, 9636–9646.
35. Furutani, Y., Shichida, Y., and Kandori, H. (2003) Structural
Changes of Water Molecules during the Photoactivation Processes
in Bovine Rhodopsin. Biochemistry 42, 9619–9625.
36. Shibata, M., Ihara, K., and Kandori, H. (2006) Hydrogen-Bonding
Interaction of the Protonated Schiff Base with Halides in a
Chloride-Pumping Bacteriorhodopsin Mutant. Biochemistry 45,
10633–10640.
37. Shibata, M., Yoshitsugu, M., Mizuide, N., Ihara, K., and Kandori,
H. (2007) Halide Binding by the D212N Mutant of Bacteriorhodopsin Affects Hydrogen Bonding of Water in the Active Site.
Biochemistry 46, 7525–7535.
38. Furutani, Y., Sudo, Y., Kamo, N., and Kandori, H. (2003) FTIR
Spectroscopy of the Complex between pharaonis Phoborhodopsin
and Its Transducer Protein. Biochemistry 42, 4837–4842.
39. Sharma, A. K., Spudich, J. L., and Doolittle, W. F. (2006) Microbial
rhodopsins: Functional versatility and genetic mobility. Trends
Microbiol. 14, 463–469.
40. Cooper, A. (1979) Energy uptake in the first step of visual
excitation. Nature 282, 531–533.
41. Birge, R. R., and Cooper, T. M. (1983) Energy Storage in thePrimary Step of the Photocycle of Bacteriorhodopsin. Biophys. J.
42, 61–69.
42. Hayashi, S., Tajkhorshid, E., and Schulten, K. (2002) Structural
Changes during the Formation of Early Intermediates in the
Bacteriorhodopsin Photocycle. Biophys. J. 83, 1281–1297.
43. Hayashi, S., Tajkhorshid, E., Kandori, H., and Schulten, K. (2004)
Role of Hydrogen-Bond Network in Energy Storage of Bacteriorhodopsin’s Light-Driven Proton Pump Revealed by ab Initio
Normal-Mode Analysis. J. Am. Chem. Soc. 126, 10516–10517.
44. Losi, A., Wegener, A. A., Engelhard, M., Gartner, W., andBraslavsky, S. E. (1999) Time-Resolved Absorption and Photothermal Measurements with Recombinant Sensory Rhodopsin IIfrom Natronobacterium pharaonis. Biophys. J. 77, 3277–3286.
45. Zhang, D., and Mauzerall, D. (1996) Volume and Enthalpy Changes
in the Early Steps of Bacteriorhodopsin Photocycle Studied byTime-Resoleved Photoacoustics. Biophys. J. 71, 381–388.
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