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Journal of Structural Biology 184 (2013) 2–11

Contents lists available at SciVerse ScienceDirect

Journal of Structural Biology
journal homepage: www.elsevier.com/locate/yjsbi

Role of trimer–trimer interaction of bacteriorhodopsin studied by optical
spectroscopy and high-speed atomic force microscopy
Hayato Yamashita a,1,3, Keiichi Inoue b,3, Mikihiro Shibata a,2, Takayuki Uchihashi a,c, Jun Sasaki b,
Hideki Kandori b, Toshio Ando a,c,⇑abcDepartment of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan
Bio-AFM Frontier Research Center, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japana r t i c l e

i n f oArticle history:
Available online 24 February 2013
Keywords:
High-speed atomic force microscopy
Bacteriorhodopsin
TrimerPoint mutation
Low-temperature UV–visible spectroscopy
Proton pumping efﬁciency

a b s t r a c t
Bacteriorhodopsin (bR) trimers form a two-dimensional hexagonal lattice in the purple membrane of
Halobacterium salinarum. However, the physiological signiﬁcance of forming the lattice has long been elusive. Here, we study this issue by comparing properties of assembled and non-assembled bR trimers
using directed mutagenesis, high-speed atomic force microscopy (HS-AFM), optical spectroscopy, and a
proton pumping assay. First, we show that the bonds formed between W12 and F135 amino acid residues
are responsible for trimer–trimer association that leads to lattice assembly; the lattice is completely
disrupted in both W12I and F135I mutants. HS-AFM imaging reveals that both crystallized D96N andnon-crystallized D96N/W12I mutants undergo a large conformational change (i.e., outward E–F loop
displacement) upon light-activation. However, lattice disruption signiﬁcantly reduces the rate ofconformational change under continuous light illumination. Nevertheless, the quantum yield of M-state
formation, measured by low-temperature UV–visible spectroscopy, and proton pumping efﬁciency areunaffected by lattice disruption. From these results, we conclude that trimer–trimer association plays
essential roles in providing bound retinal with an appropriate environment to maintain its full photoreactivity and in maintaining the natural photo-reaction pathway.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction
Intermolecular interactions play a pivotal role in the function of
membrane proteins. Numerous membrane proteins interact witheach other or other components in two-dimensional membrane
environments, e.g., self-assembly into oligomers or crystals (Essenet al., 1998; Persike et al., 2001; van Huizen et al., 1999; Colomet al., 2012), receptor-mediated protein–protein interactions for signal transduction (Szidonya et al., 2008), and the membrane localization of proteins via association with lipid rafts (Simons and Ikonen,

Abbreviations: AFM, atomic force microscopy; bR, bacteriorhodopsin; PM,
purple membrane; HPLC, high-performance liquid chromatography; HS-AFM,
high-speed atomic force microscopy; WT, wild-type.
⇑ Corresponding author at: Department of Physics, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan. Tel.: +81 76 264 5663; fax: +81 76
264 5739.
E-mail address: tando@staff.kanazawa-u.ac.jp (T. Ando).
1
Present address: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
2
Present address: Department of Neurobiology, Duke University Medical Center,North Carolina, USA.
3
These authors contributed equally to this work.
1047-8477/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jsb.2013.02.011

1997). Thus, elucidating the intermolecular packing of membraneproteins is essential for understanding the structure–function relationship and the general principle of membrane protein assembly.
Bacteriorhodopsin (bR), a light-driven proton pump in Halobacterium (H.) salinarum (Oesterhelt and Stoeckenius, 1971), is one of
the best-characterized transmembrane proteins (see reviews: Haupts et al., 1999; Lanyi, 2004; Lanyi, 2006; Hirai et al., 2009). Under
the native condition, bR forms trimers that assemble into a twodimensional hexagonal lattice called the purple membrane (PM)(Blaurock and Stoeckenius, 1971). Previously, numerous structural
studies of bR in PM were performed under unphotolyzed (Sasset al., 2000) or frozen activated (Luecke et al., 1999; Subramaniamand Henderson, 2000; Vonck, 2000; Lanyi and Schobert, 2003; Hirai and Subramaniam, 2009) states. Recently, by means of highspeed atomic force microscopy (HS-AFM) (Ando et al., 2001; Andoet al., 2008), light-induced conformational changes of bR have beenvisualized in real-time and real-space (Shibata et al., 2010; Shibata
et al., 2011). HS-AFM movies have revealed that the E–F inter-helical loop of each bR monomer moves outwards from its trimer center upon photo-excitation from the ground state to the MN state,
which results in contact among three nearest-neighbor bR monomers, each belonging to a different adjacent trimer. This triad ofAuthor's personal copy

H. Yamashita et al. / Journal of Structural Biology 184 (2013) 2–11

nearest-neighbor monomers is designated the ‘trefoil’. This bR–bR
interaction within a trefoil, which occurs under a relatively strong
light illumination, engenders both positive and negative (i.e., bipolar) cooperative effects in decay kinetics; an early activated monomer within a trefoil decays more slowly, whereas the latest
activated monomer within a trefoil decays faster than that in thecase in which only one monomer within a trefoil is activated(and hence no bR–bR interaction occurs) under weak light illumination. However, because of bipolar cooperative effects, the overallturnover rate is maintained over a wide range of light intensities(Shibata et al., 2010).
An HS-AFM observation has also showed that a bR trimer occasionally rotates around a pivotal point at the interface between
dense crystal and non-assembled areas of PM, suggesting the presence of speciﬁc inter-trimer interaction sites at the outer rim of abR trimer (Yamashita et al., 2009). This is consistent with the result
obtained from a neutron diffraction study suggesting that some of
the aromatic residues (W10, W12, Y131, and F135) located vertically no farther than 5 Å into the extracellular side of the membrane and laterally at the outer rim of a trimer are involved inlattice formation (Weik et al., 1998). This suggestion is derived
from the measured glycolipid distribution in the membrane andthe possible hydrophobic stacking between aromatic side chainsand carbohydrates (Qian et al., 1995) as well as from the close
proximity between W10–W12 and Y131–F135 in the bR assembly.
However, a more deﬁnite and speciﬁc identiﬁcation of amino acids
responsible for lattice formation has not been performed eversince.
In the present study, we speciﬁed amino acid residues involvedin trimer–trimer association by preparing ﬁve bR mutants in whichisoleucine was substituted for the aromatic residues mentioned
above or phenylalanine was substituted for W12. HS-AFM observations of these mutants showed that the trimers of bR mutants(W12I and F135I) do not assemble and instead rapidly diffuse in
the membrane. Furthermore, the HS-AFM observation of the
D96N/W12I bR mutant revealed that lattice disruption reduces
the rate of light-induced conformation change, which is proportional to light intensity. Interestingly, the spectroscopic and proton
pumping assay studies, however, showed that proton pumping
efﬁciency is unaffected by W12I mutation. Taking these results
into consideration, we propose a model in which lattice disruption
causes the photo-reaction pathway to branch into two: the original
productive pathway and a new non-productive pathway. In the
non-productive pathway, no signiﬁcant structural change occurs
in bR and no proton is pumped out; however, the kinetics on thispathway is much faster than that on the original pathway. Thismodel quantitatively well explains all the results obtained in thisstudy. Thus, trimer–trimer association plays essential roles in providing bound retinal with an appropriate environment to maintainits full photo-reactivity and in maintaining the natural photo-reaction pathway.

2. Materials and methods
2.1. Plasmid constructs and site-directed mutagenesis
A template plasmid containing the bop gene (1.6 kbp) cloned
into the vector pGEM-T easy (Promega) was used (Shibata et al.,2007). Point mutations were introduced into the bop gene by
PCR mutagenesis (Stratagene) as follows; W10I, Trp10 ? Ile;
W12I, Trp12 ? Ile; Y131I, Tyr131 ? Ile; F135I, Phe135 ? Ile;
W12F, Trp12 ? Phe; D96N, Asp96 ? Asn; and D96N/W12I, Asp96 ? Asn and Trp12 ? Ile. The mutated fragments were transferred to the vector pMPK69 for transformation (Peck et al.,2000), which contains a gene conferring resistance to the 3-hydro-3xy-3-methylglutaryl CoA reductase inhibitor (simvastatin, Wako)on H. salinarum. The resultant mutations were conﬁrmed bysequencing the plasmids prior to the transformation of H.
salinarum.
2.2. Transformation of H. salinarum and puriﬁcation ofbacteriorhodopsin mutants
H. salinarum strain MPK409 (Peck et al., 2000) was transformed
using the plasmids described above. The transformation procedure
was essentially as described previously (Cline and Doolittle, 1987).
Brieﬂy, 10 mL of cells in the early logarithmic growth phase wascollected by centrifugation (6000g, 10 min) and resuspended in
500 lL of a spheroplasting solution to facilitate plasmid DNA transfection into the cells. 200 lL-aliquots of the suspension wereadded to 10 lL of 0.5 M EDTA in a spheroplasting solution andgently mixed. After 20 min, 15 lL of a plasmid DNA solution wasadded, and the resulting solution was incubated for 5 min. To thissolution, an equal volume (225 lL in this case) of 60% polyethylene
glycol 600 in a spheroplasting solution was added; the solution obtained was mixed gently by back and forth tilting of the tube. After
20 min of additional incubation, 10 mL of a regeneration salt solution containing 15% sucrose was added. Cells were pelleted by
microcentrifugation at 5000 rpm for 20 min, and 10 mL of a regeneration salt solution was again added to the resultant pellet. Afterincubation for 24 h at 37 °C, 100-lL samples were spread on agar
plates containing 15% sucrose and 10 lg/mL simvastatin. After10 days of incubation at 37 °C, purple colonies were picked andgrown in a complex culture medium with 10 lg/mL simvastatin.
After incubation for 24 h at 37 °C, 100-lL samples were spreadon agar plates containing 250 lg/mL 5-ﬂuoroorotic acid (Wako).
After a week of incubation, purple colonies were picked and grown
again in a complex culture medium containing 50 lg/mL uracil, as
uracil cannot be synthesized by the transformed cells lacking the
URA3 gene coding for orotine-5-monophosphate decarboxylasethat converts 5-ﬂuoroorotic acid into toxic 5-ﬂuorouracil. The purple membranes were isolated and puriﬁed by a usual method(Oesterhelt and Stoeckenius, 1974). As W12I, F135I and D96N/
W12I do not form 2D crystals in the native membrane, the bottom
layer containing the highest bR content was collected by sucrose
density gradient centrifugation. After removing sucrose by sedimentation, the pellets were suspended in a solution (Buffer-A) containing 10 mM phosphate (pH 7.0) and 300 mM KCl. For AFM
observation, the samples were diluted to 0.1 mg/mL in Buffer-A.
2.3. HS-AFM observation
The laboratory-built HS-AFM apparatus used in this study issimilar to that previously reported (Ando et al., 2008). The detailed
procedures towards HS-AFM imaging are reported elsewhere(Uchihashi et al., 2012). HS-AFM images were acquired in the tapping mode. To detect cantilever deﬂection, we used an opticalbeam deﬂection detector equipped with an infrared laser(980 nm) to avoid exciting bR. The laser beam was focused ontoa small cantilever using a 50 objective lens. The cantilever witha spring constant of 0.2 N m1 (Olympus) is 6–7 lm long, 2 lm
wide and 90 nm thick. Its resonant frequency and quality factor
in an aqueous solution are 1 MHz and 2, respectively. An amorphous carbon tip was grown on the original cantilever tip by electron beam deposition. Tip length was adjusted to 1 lm. The tip
apex was sharpened by plasma etching under argon gas (4 nm
in radius). For HS-AFM imaging, the cantilever free-oscillation
amplitude was set at 1 nm, and the set-point amplitude was set
at 90% of the free-oscillation amplitude. A sample stage made ofquartz glass was placed on the z-scanner, and a 1.5-mm-diametermica disk was glued onto the sample stage. A 2-lL sample droplet

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was deposited on the freshly cleaved mica surface and then incubated for 3 min. In the HS-AFM observation of the non-assembledbR samples, bR was chemically anchored on a substrate surface to
avoid its rapid diffusion. For this purpose, we used a mica surface
treated with 3-aminopropyltriethoxysilane (aminosilane) (ShinEtsu Silicones) and then with glutaraldehyde (Nacalai tesque);ﬁrst, 0.1% aminosilane solution in water was placed on a freshly
cleaved mica surface and incubated for 3 min. After rinsing withwater, a 0.1% glutaraldehyde solution was placed on the mica surface and incubated for 3 min. After rinsing the surface with water, asample droplet was placed on the mica surface and incubated for
3 min. HS-AFM measurement was performed under Buffer-A solution at room temperature.
For the photo-excitation of bR samples, a green laser (532 nm)
was irradiated through the 50 objective lens. The intensity measured at the exit of the objective lens was varied in the range of
0.02–1 lW.
The center of mass of each imaged bR monomer was analyzed
as reported previously (Shibata et al., 2010). First, a whole AFM image was segmented into each monomer region using a watershedalgorithm. Second, the grayscale of each segment was digitizedusing a threshold determined by a fuzzy-entropy algorithm. At
each segment, an ensemble of pixels with grayscales above thethreshold was deﬁned as a monomer region (region of interest,
ROI). Finally, the center of mass of a monomer is calculated using
the X, Y and Z coordinates of pixels in the ROI.
2.4. UV–visible spectroscopy
The absorption spectra of bR mutants were measured at room
temperature using a UV–visible spectrometer UV-1700 (Shimadzu). The bR samples were suspended in a solution containing
10 mM phosphate (pH 7.0) and 300 mM KCl.
2.5. HPLC analysis
A high-performance liquid chromatography (HPLC) analysis of
the composition of retinal isomers was conducted as previously
described (Shimono et al., 2001). The HPLC apparatus was
equipped with a silica column (6.0 ø  150 mm3; YMC-Pack SIL).
The solvent composed of 12% (v/v) ethyl acetate and 0.12% (v/v)
ethanol in hexane was supplied at a ﬂow rate of 1.0 mL/min. The
extraction of retinal oxime from the bR samples was carried out
in hexane after denaturation by methanol and 500 mM hydroxylamine at 4 °C. The molar composition of retinal isomers was calculated from the peak area corresponding to each retinal isomer inthe HPLC elution patterns. The assignment of the peaks was performed as reported previously (Sudo et al., 2011). Three independent measurements were averaged.

absorbance of both samples was adjusted to 0.6 at 580 nm to makethe number of absorbed photons equivalent. The photo-reaction
rate of D96N/W12I bR relative to that of D96N bR was estimatedby the comparison of the magnitudes of absorption change (corresponding to the accumulation of M-intermediate) under identical
illumination conditions.

2.7. Proton pumping activity assessment
H. salinarum strain MPK409 cells expressing either wild-type(WT) bR or W12I bR were grown up to the stationary phase(>1 week) in a complex culture medium at 37 °C with aerationand illumination. 100 mL of the culture was washed by centrifugation at 5000 rpm followed by the suspension of the cell pellet in abasal salt solution. After repeating the wash cycle three times, thecell pellet was suspended in 10 mL of a basal salt solution. The cellsuspension was then transferred to a glass container whose temperature was regulated by circulating water adjusted at 20 °C.
The sample was stirred throughout the equilibration and recording. Illumination light was provided by a 1000 W halogen-tungsten
lamp output (HILUX-H130, Rikagaku Seiki, Japan) directed to thesample through a yellow cutoff ﬁlter (Y52, Toshiba, Japan). The
pH of the sample was measured with a pH probe (9621-10D, HORIBA, Japan) and a pH meter (F-55, HORIBA, Japan). The pH changes
occurring in the medium during bR photoactivation were calibrated by pH changes induced upon the addition of 5 lL of
100 mM HCl to the cell suspension (50 lM in ﬁnal concentration).
In the above measurement of pH change, the observed pHchange was calibrated by the amount of bR in each cell suspension.
To determine the amount of bR, 10 mL of the sample was disrupted
by sonication. The membrane fraction was isolated according to
the standard procedure (Oesterhelt and Stoeckenius, 1974) andsuspended in 2 mL of 50 mM Tris–HCl at pH 7.5. The amount of
WT bR in the solution was assessed from the change in the absorption spectrum of bR caused by complete photobleaching. The
photobleaching was carried out in the presence of 250 mM NH2OH
under light illumination for 10 min at ambient temperature. The
light was provided in the same way as in the pH measurement described above. In WT bR, the molar extinction coefﬁcient of
63,000 M1 cm1 at 570 nm was used for estimating molar content. To obtain the amount of W12I bR whose molar extinction
coefﬁcient is unknown, the change in its absorption at 350 nm
caused by complete photobleaching was compared with that of
WT bR because the absorption at 350 nm is proportional to theamount of retinal oxime produced by photobleaching.

3. Results and discussion

2.6. Low-temperature UV–visible spectroscopy

3.1. High speed AFM observation of bR mutants

The photo-reaction efﬁciency of D96N/W12I bR relative to
D96N bR was estimated from the changes in the UV–visible
absorption spectra of both samples upon light illumination at
210 K. For this estimation, the absorption spectra were measured
using a UV–visible spectrometer (V-550, JASCO, Japan) equippedwith a cryostat (DN1704, Oxford Instruments, UK). The sample
was placed on a BaF2 window of 18 mm diameter, and a dried
PM ﬁlm was hydrated by placing a drop of H2O next to the ﬁlm.
The hydrated ﬁlm on the BaF2 window was covered with anotherwindow and placed in the cryostat sample cell in the spectrometer.
The photo-reaction of the sample was initiated by the illuminationof 580 ± 10 nm light from a halogen-tungsten lamp (1000 W) (HILUX-H130, Rikagaku Seiki, Japan) for total 300 s at 210 K, and thespectral changes were measured at 10-s intervals. The initial

Fig. 1A shows a schematic of the bR lattice assembly constructed from the PDB (1BRR) structure of bR trimers (Essenet al., 1998). The positions of W12 and F135 are highlighted by
the white and light-blue spheres, respectively. The measurementof gycolipid distribution in PM by previous neutron diffraction
studies suggested that some of the aromatic residues (W10, W12,
Y131, and F135), which locate vertically no farther than 5 Å into
the extracellular side of the membrane and laterally at the outerrim of the trimer, participate in inter-trimer association (Weiket al., 1998). Our previous high-resolution AFM images also suggestthat the trimer–trimer association sites locate at or near the W12
residue (Yamashita et al., 2009). To identify amino acid residues
responsible for inter-trimer association more speciﬁcally, we constructed ﬁve bR mutants (W10I, Y131I, W12I, F135I, and W12F).

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5

Fig. 1. (A) Schematic of bR lattice assembly (PDB 1BRR). The positions of W12 and F135 in the bR model are indicated by the white and light-blue spheres, respectively. (B–F)
Absorption spectra of prepared bR mutants. (G–K) HS-AFM images of cytoplasmic surfaces of bR mutants. Images in G, H and K clearly show a packed arrangement of bR
molecules. bR trimers are arranged in a hexagonal lattice with unit cell dimensions of 62 Å. Imaging rates: (G, H and K) 1 frame/s, (I and J) 3 frames/s. Z-scales: (G) 2.0 nm, (H)
2.4 nm, (I) 7.3 nm, (J) 4.0 nm, (K) 1.6 nm.

Fig. 1B–F shows the absorption spectra of the PM samples, eachcontaining one of the ﬁve bR mutants. All of these spectra show theabsorption bands around 550 nm, indicating that all mutants havea light sensitive retinal chromophore. AFM images of these bR mutants are shown in Fig. 1G–K. As can be seen in these AFM images,
the W10I and Y131I mutants form a lattice assembly (Fig. 1G and
H). The conﬁguration of these lattices is identical to that of WT bR
reported previously (Yamashita et al., 2009). In contrast, the latticeassembly is not seen in the AFM images of the W12I and F135I mutants (Fig. 1I and J), but some isolated trimers are seen, as indicatedby dotted triangles in the images. The isolated trimers quickly diffused within the native membrane (see Movie S1 in Supporting
Materials). These results indicate that both W12 and F135 residues
play an essential role in assembling bR trimers (see Fig. 1A). On theother hand, the W12F mutant forms a lattice assembly, as shown in
Fig. 1K. This indicates that aromatic residues at positions 12 and
135 associate with each other possibly through a hydrophobic
stacking interaction mediated by the sugar moieties of glycolipids
contained in the membrane (Qian et al., 1995; Weik et al., 1998).
3.2. Light-induced conformational change of bR in isolated trimer
We have recently succeeded in visualizing the light-induced conformational change of the D96N bR mutant (Shibata et al., 2010)
using HS-AFM. While the wild type completes the photocycle within10 ms, this mutant does it within 10 s at pH 7 but retains its proton pumping ability. This long photocycle facilitates the HS-AFM

observation of the photo-induced conformational changes of bR.
To investigate the conformational change of bR in isolated trimers
under light illumination, the mutation D96N was introduced to
W12I bR. D96N/W12I bR also did not assemble into a lattice(Fig. 2B). Since non-assembled bR trimers diffuse on a bare mica surface too rapidly to be imaged even by HS-AFM, D96N/W12I bR trimers were immobilized on a mica surface chemically modiﬁedwith aminosilane and glutaraldehyde (Fig. 2A). Fig. 2B shows the
non-assembled bR trimers immobilized on the mica surface.
Fig. 2C shows successive AFM images of a D96N/W12I trimer, captured at 2 frames/s (also see Movie S2 in Supporting Materials). Under green light illumination, bR molecules in the trimer appeared tomove outwards from the trimer center owing to the outward movement of the corresponding E–F loops (compare the image at 0 s withthose at 1–4 s in Fig. 2C); they subsequently returned back to theirinitial positions within a few seconds after light was switched off.
However, we noticed the presence of partially inactive trimers;
one or two bR molecules within a trimer did not exhibit discernible
outward displacement even under long illumination (35% of theexamined bR molecules). The presence of partially inactive trimers
was in contrast to the case of assembled D96N bR trimers where illuminated molecules mostly appeared to move outwards. Fig. 2Dshows the displacement of the center of mass as a function of time
for three bR monomers within a full-active D96N/W12I trimer(M1–M3 in Fig. 2D). The intermediate state of D96N with the excitedconformation has been designated MN (Kamikubo et al., 1996; Sasaki
et al., 1992) because it exhibits an outward tilt of helix F similar toAuthor's personal copy

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H. Yamashita et al. / Journal of Structural Biology 184 (2013) 2–11

Fig. 2. (A) Schematic of anchoring bR onto chemically modiﬁed mica surface. (B) HS-AFM image of D96N/W12I bR trimers anchored on mica substrate. (C) Clips of
successively captured HS-AFM images of bR trimer anchored on substrate. The green bars indicate the illumination of 532-nm green light of 0.2 lW. The insets are schematic
illustrations of the observed bR conformations. The gray and green circles indicate the conformations of bR monomers in the ground and excited states, respectively. Imaging
rates: (B) 1 frame/s, (C) 2 frames/s. Z-scales: (B) 3.7 nm, (C) 1.6 nm. (D) Displacement of the center of mass as a function of time for three bR monomers (M1–M3) within a full
active D96N/W12I trimer. The green regions show periods of green light illumination (532 nm).

that of the N state of WT (Vonck, 1996), while the Schiff-base is still
deprotonated like the precedent M state owing to the absence of the
proton donor residue (Asp96) for the Schiff base. The conformationalchange is reproducible throughout the repeated dark-illumination
cycles (Movie S2). The observed behaviors of the double mutant
are similar to those observed previously for the D96N mutant (Shibata et al., 2010), except for the partial presence of photo-inactivebR molecules in the D96N/W12I sample (to be discussed later).

3.3. Analysis of reaction kinetics and its comparison between D96N bR
and D96N/W12I bR
We analyzed more details of the photo-induced conformationalchanges of bR to investigate a possible effect of lattice disruption
on the photo-activated reaction kinetics. We ﬁrst analyzed the timecourse of photo-activation under continuous light illumination.
Fig. 3A shows the time courses in which the population of activatedAuthor's personal copy

H. Yamashita et al. / Journal of Structural Biology 184 (2013) 2–11

D96N and D96N/W12I bR molecules increased after continuous illumination was started. The rise time constant of D96N/W12I bR was
estimated to be s = 1.9 ± 0.06 s, which was four fold longer than thatof D96N bR (s = 0.49 ± 0.01 s) under the same light intensity. On theother hand, as shown in Fig. 3B, the time constants of decay after
light-off were almost identical for both mutants (s  10 s). To ruleout the possibility that the rise delay of D96N/W12I bR is causedby the anchoring of the molecules onto the chemically modiﬁed
mica, we also observed D96N bR attached onto the chemically modiﬁed mica and examined the time constants in the rise and decay
phases. As shown in the insets of Fig. 3A (rise phase) and Fig. 3B (decay phase), the respective time constants were essentially identicalto those of D96N bR simply placed on a bare mica surface. Moreover,
all the molecules under illumination appeared to move outwards.
Thus, the chemical anchoring has no signiﬁcant effect on the photocycle. The rise delay and the presence of inactive molecules observedwith D96N/W12I bR are intrinsically caused by lattice disruption.
We previously found that the decay kinetics after the light-off ofthe photo-induced conformational change in D96N bR differs
depending on the intensity of illuminated light (Shibata et al.,2010). When the intensity is strong (0.5 lW at the exit of theobjective lens; likewise, the light intensities indicated below arethose measured at the exit of the objective lens), the decay rateof many excited molecules splits into shorter and longer ones
(2 s vs. 13 s) relative to the rate (7 s) observed with rarely excited molecules under weak illumination (0.01 lW). We concluded that this altered decay kinetics is caused by theinteraction between bR molecules that are excited together not

7

within a trimer but within a ‘trefoil’. Earlier works have also reported that the spectroscopically measured kinetics of the bR
photocycle is altered depending on light intensity, and attributed
the observed cooperative effects to neighboring effects within trimers (Rehorek and Heyn, 1979;Tokaji, 1993) or general crowding
effects in the lattice (e.g., Varo et al., 1996). However, the cooperative effects observed in these ensemble-averaged spectroscopic
measurements are not as distinct as those observed by HS-AFM,
which is due likely to the bipolar nature of the cooperativity. Here,the issue of the cooperativity origin is further certiﬁed by examining the decay kinetics of D96N/W12I bR in full active trimers, as its
trimers are isolated from each other in the membrane (i.e., absenceof trefoil). Fig. 3C shows the decay of rarely activated D96N molecules under 0.022 lW illumination. The decay time is almost identical to that measured for D96N/W12I bR under 0.2 lW
illumination (blue line in Fig. 3B; s  10 s). Next, we measured
the decay kinetics of activated monomers within full-active
D96N/W12I trimers under stronger illumination (up to 1 lW). In
this measurement, we analyzed the decay kinetics separately forearly-activated (red circles in Fig. 3D) and late-activated monomers (blue circles in Fig. 3D) within each D96N/W12I trimer. The
separately analyzed decay time constants were almost identical(s  10 s). Thus, the disruption of lattice assembly in the W12IbR mutant eliminates the light-intensity dependence of the decay
kinetics observed with lattice-assembled bR, reinforcing our previous conclusion that the bipolar cooperative effects in the decay
kinetics are engendered by the interaction between excited bR
molecules within a trefoil (not within a trimer).

Fig. 3. (A) Time courses of accumulation of activated bR molecules detected by HS-AFM under continuous light illumination (0.2 lW). Red plots, unanchored D96N bR; blue
plots, anchored D96N/W12I bR. The exponential time constants (s) of D96N and D96NW12I are 0.49 ± 0.01 s (total number of analyzed bR molecules, NT = 51; number ofdifferent molecules analyzed, Nm = 22) and 1.9 ± 0.06 s (NT = 51, Nm = 16), respectively. The inset shows the time course of the accumulation of activated bR in an anchored
D96N bR sample. The exponential time constant (s) is 0.58 ± 0.01 s. (B) HS-AFM-detected decay of activated states of unanchored D96N bR (red plots) and anchored D96N/
W12I bR in full active trimers (blue plots) after light (0.2 lW) was switched off. The exponential time constant (s) of D96N and D96NW12I are 10.4 ± 0.10 s (NT = 51, Nm = 22)
and s = 9.6 ± 0.04 s (NT = 51, Nm = 16), respectively. The inset shows the decay of activated molecules for anchored D96N after light (0.2 lW) was switched off. The
exponential time constant (s) is 9.3 ± 0.04 s. (C) Decay of activated molecules for unanchored D96N after weak light (0.022 lW) was switched off. As shown in the inset, under
this weak illumination, only one monomer (red circle) is excited in a trefoil, while the other two monomers (the grey circles) in the same trefoil are not excited. The
exponential time constant (s) is 9.1 ± 0.12 s. (NT = 51, Nm = 30) (D) Decay of activated D96N/W12I bR monomers in full active trimers after strong light illumination (0.2–
1.0 lW) was switched off. The decay analysis was made separately for early-activated (red plots) and late-activated (blue plots) monomers within a trimer, as depicted by theinset illustration (red circles, early-activated molecules; blue circles, late-activated molecules). The number in these circles indicates the order in which molecules areactivated. The exponential time constants (s) for the early- and late-activated molecules are 12.2 ± 0.31 s (NT = 20, Nm = 30) and 9.9 ± 0.18 s (NT = 20, Nm = 34), respectively.

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3.4. HPLC analysis of retinal in D96N and D96N/W12I mutants
The introduction of W12I mutation to D96N bR signiﬁcantly retards the accumulation of the MN state (outward E–F loop displacement) under continuous illumination, compared with that of thelattice-assembled D96N bR (Fig. 3A). Since the rise rate linearly increases with illumination intensity (Fig. 4A), the retardation is
likely attributable to lower efﬁciency of the conformational changeor to a smaller optical absorption cross section of the D96N/W12I
mutant. The latter is, however, ruled out because the difference
spectrum between the ground and 100%-excited states of D96N/
W12I bR measured at low temperature (210 K) was identical tothat of D96N bR, as shown in Fig. 6. Therefore, the reduced accumulation rate should be attributed to reduced efﬁciency of the conformational change of D96N/W12I bR. This suggests that there is asecondary pathway in which light-adsorbed D96N/W12I bR does
not proceed to the MN state (to be discussed later).
A previous AFM imaging study on native PM showed thatphotobleaching in the presence of hydroxylamine (Moller et al.,2000) causes bR lattice disassembly, suggesting that the removalof the retinal chromophore from bR weakens inter-trimer association. It is therefore conceivable that the loss of inter-trimer association due to W12I mutation leads to an alteration of theenvironment around retinal in bR, which might be involved in reduced efﬁciency of the conformational change of D96NW12I bR
and/or the presence of inactive molecules. To investigate thispossibility, the isomeric compositions of retinal in D96N and

D96N/W12I and their quantum yields of M-formation werecompared.
HPLC analysis was performed for retinal extracted from D96NbR and D96N/W12I bR in hydroxylamine. Fig. 5A shows chromatograms of retinal isomers extracted from each bR mutant under the
dark- and light-adapted conditions. The retinal isomer compositions calculated from the chromatograms are summarized inFig. 5B and Table 1. The retinal composition of D96N bR is similar
to that of WT bR, that is, almost equal amounts of the all-trans and13-cis isoforms exist in the dark, whereas the all-trans form predominates (93%) over 13-cis after light adaptation (Pettei et al.,1977). In contrast, D96N/W12I bR shows a reduction in the degreeof difference in isomeric composition between the light- and darkadapted states (64% all-trans and 32% 13-cis in the dark-adapted
state; 68% all-trans, 23% 13-cis, and trace amounts of 9-cis and11-cis in the light-adapted state). Since the HS-AFM measurement
was carried out after the light adaptation of the samples, D96N bR
and D96N/W12I bR contain 93% and 68% all-trans retinal, respectively. Consistent with this analysis, 36.7% of the D96N/W12I bR
molecules (among 147 molecules in total) observed by HS-AFM
showed no discernible structural changes upon light illumination.
Those molecules are considered to contain 13-cis retinal. However,this does not account for the retarded accumulation of the MN state
observed under constant light illumination.
3.5. Comparison between D96N bR and D96N/W12I bR by lowtemperature UV–visible spectroscopy
The reduced efﬁciency of the conformational change of D96N/
W12I bR is likely to be caused by an alteration in the photochemical reaction. Here, we compared the relative quantum yields ofM-formation in D96N bR and D96N/W12I bR using UV–visible
spectroscopy at low temperature (210 K) where the M state isabsolutely stable (Sasaki et al., 1992). The hydrated ﬁlms of thebR mutants were illuminated with 580-nm light. The spectra were
measured after every 10-s illumination and the illumination was
performed for 300 s in total (Fig. 6). The accumulation of the Mintermediate and the depletion of the initial state were estimatedfrom the corresponding absorption change at 415–420 nm and575–580 nm, respectively. The blue plots in Fig. 6C and D show
the time courses of M-accumulation in D96N bR and D96N/W12I
bR, respectively. The initial absorption peaks at the excitation
wavelength (580 ± 10 nm) are the same for both samples. Therefore, both samples could be excited with the same efﬁciency,
allowing relative comparison of the quantum yields of M-formation by directly comparing the time courses shown by the blue
plots in Fig. 6C and D. The time-constant thus obtained for
D96N/W12I bR (s = 135 s) was 1.5-fold smaller than that obtainedfor D96N bR. This means that the quantum yield of M-formation in
D96N/W12I bR is reduced by 30% compared with that in D96N
bR. This reduced quantum yield should be explained by the 30%
reduction in the amount of all-trans retinal, as revealed by the
HPLC analysis. This is because bR with 13-cis retinal has beenshown to produce no M state with the deprotonated Schiff-base(Gergely et al., 1994). However, this result again does not accountfor the retarded accumulation of the MN state observed with D96N/
W12I bR under constant light illumination.
3.6. Measurement of proton pumping efﬁciency

Fig. 4. Rate constants of rise (A) and decay (B) phases as functions of light intensity
for D96N (red plots) and D96NW12I (blue plots) measured by HS-AFM. These rate
constants were ﬁtted with linear functions with slopes of 6.1 ± 0.43 (red line) and
1.5 ± 0.15 (blue line) for the rise phase, and with slopes of 0.02 ± 0.01 (red line) and0.007 ± 0.01 (blue line) for the decay phase. The insets show the rate constants of
the rise (A) and decay (B) phases as functions of light intensity for D96N measured
by UV–visible spectroscopy.

If helix-F displacement is tightly coupled with (or prerequisitefor) unidirectional proton pumping, the reduced yield of the conformational change of D96N/W12I bR would result in a reduced
proton pumping efﬁciency, compared with that of D96N bR. To address this issue, we monitored the proton pumping activity of bR
molecules by measuring light-induced pH changes in the medium

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H. Yamashita et al. / Journal of Structural Biology 184 (2013) 2–11

9changes because of their slow photocycle). To compare their
pumping activities, light-induced changes in the proton concentration were normalized by the respective bR amounts (Fig. 7). Notethat this proton pumping assay can only detect the pumping activity in the steady state because the photocycle of WT bR and W12IbR is very fast. Pumping efﬁciency was evaluated from the initialslope of the proton concentration increase after the onset of illumination with >500-nm light. The estimated number of protons
pumped per one bR molecule was 15–18/min for both WT bR
and W12I bR. Likewise, the amplitude of the proton concentration
change was comparable for both samples (Fig. 7). Thus, the introduction of W12 mutation does not appear to affect proton pumping activity.
3.7. Relationship between E–F loop movement and proton pumping

Fig. 5. Composition analysis of retinal isomers in D96N and D96N/W12I. (A) HPLC
patterns of retinal extracted from D96N under dark-adapted state (blue) and after
illumination of light (cyan), and from D96N/W12I under dark adapted state (darkgreen) and after illumination of light (light green). (B) Compositions of retinal
isomers extracted from D96N and D96N/W12I under dark- and light-adapted
conditions. For light adaptation, light of >500 nm wavelength was illuminated for
1 min.

in which H. salinarum cells expressing WT bR or W12I bR were suspended (although we should have used D96N bR and D96N/W12I
bR for this assay, it was difﬁcult to measure light-induced pH

The present HS-AFM and spectroscopic studies gave the following results: (i) the MN state with an outwardly displaced E–F loop
exists in the photocycle of D96N bR and D96N/W12I bR; (ii) after
the start of continuous illumination, the MN state accumulationof disassembled D96N/W12I bR occurs with a rate 4-times lower
than that of assembled D96N bR; (iii) the accumulation rate of both
samples linearly increases with increasing light intensity; (iv) the
optical absorption cross section is very similar between D96N bR
and D96N/W12I bR; and (v) the introduction of W12I mutation
into WT bR does not appear to affect the proton pumping activityof bR within experimental accuracy. From these results, we propose that the photo-reaction cycle is altered by the introductionof W12I mutation, as shown in Fig. 8. The unspeciﬁed MX state occurs ﬁrst upon photo-excitation, after which the pathway branches
into two: in one pathway, the MN state is formed, while in theother pathway, the MX state is de-excited to the ground state G. Results of numerical analysis based on this proposed reaction scheme
are shown in Fig. 9. The retardation of MN state formation by the
introduction of W12I mutation (compare the red and blue solidlines in Fig. 9A) as well as the proportionality of MN state accumulation rates to light intensity (Fig. 9B) are similar to those observed
experimentally. The W12I mutation signiﬁcantly reduces theground-state depletion rate (compare the red and blue dotted lines

Fig. 6. Comparison of photo-reactivities between D96N and D96N/W12I. The absorption changes of (A) D96N and (B) D96N/W12I after the irradiation of light (580 ± 10 nm)
are shown for the indicated irradiation durations (10–300 s). The blue squares indicate the degrees of accumulation of M intermediate monitored at 415 nm for D96N (C) andat 420 nm for D96N/W12I (D). The green circles indicate the bleaching of the initial state monitored at 577 nm for D96N (C) and at 581 nm for D96N/W12I (D).

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Table 1
Compositions of retinal isomers extracted from D96N and D96N/W12I under the dark and light adapted conditions.
Retinal isomer

D96N Dark

D96N Light

D96NW12I Dark

D96NW12I Light

All-trans
13-cis9-cis
11-cis50.5 ± 0.8%
49.5 ± 0.8%
0%0%93.5 ± 0.8%
6.4 ± 0.8%
0%0%64.3 ± 0.3%
31.7 ± 0.4%
2.45 ± 0.11%
1.6 ± 0.2%

68.3 ± 0.9%
23.0 ± 0.8%
5.2 ± 0.3%
3.55 ± 0.19%

Fig. 7. Measurement of proton pumping efﬁciency. Proton pumping activity wasmonitored by the measurement of light-induced (>500 nm) pH changes in a basal
salt solution in which H. salinarum strain MPK409 expressing either WT (blue line)or W12I (purple line) was suspended. The initial pH in the dark was 6.5. The time
points for light-on and light-off are indicated by the downward arrows and the
upward arrows, respectively. The proton concentration changes were normalized
by the protein concentrations and calibrated by the measurement of pH changes
induced by the addition of 50 lM HCl.

Fig. 8. Models of photocycles in D96N bR and D96N/W12I bR. Upper illustrations
indicate the lattice-assembled D96N bR (left) and non-assembled D96N/W12I bR(right). The bottom schematics show the modeled photocycles in D96N bR (left) andD96N/W12I bR (right). In the photocycle of D96N bR, the presence of a direct
recovery pathway from MX with no E–F loop displacement is minor as indicated by
the dashed arrow. The rate constants, k1, k2, k3, and k4, for transitions between
different states are assigned as shown in the right bottom schematic.

in Fig. 9A); however, the spectroscopically measured rates arenearly identical between the D96N/W12I and D96N mutants(shown by curves with green circles in Fig. 6C and D). However,this apparent discrepancy is explained by the low temperatureused for the spectroscopic measurement (i.e., the M state is completely stabilized and likewise the MX state is likely to be stabilized
to some extent at low temperature). In the steady state (t > 10 s),
the time course of proton accumulation by D96N/W12I is only
slightly lower (15%) than that by D96N (Fig. 9C). This accumulation

Fig. 9. Numerical analyses of MN formation rate and proton pumping activity basedon the modeled photocycle in D96N bR and D96N/W12I bR depicted in Fig. 8. (A)Time courses of MN state accumulation (solid lines) and ground state depletion(dotted lines) for D96N bR (red lines) and D96N/W12I bR (blue lines) after
continuous light illumination. The time courses of MN state accumulation arenormalized by respective saturated values. (B) Relationship of MN state accumulation rate and light intensity for D96N bR (red line) and D96N/W12I bR (blue line).
(C) Time courses of proton accumulation by D96N bR (red line) and D96N/W12I bR(blue line). Here, it was assumed that proton pumping only occurs through MN. In
the calculations of (A) and (C), the following rate constants (see Fig. 8) were used:
k1 = 2.04 s1, k2 = 100.0 s1, k3 = 0.1 s1, and k4 = 0 s1 (D96N bR) or 370.4 s1(D96N/W12I bR). In the calculation of (B), the rate constant k1 was varied from 0 s1
to 12.24 s1, while the other rate constants were the same as those used for (A) and(C).

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H. Yamashita et al. / Journal of Structural Biology 184 (2013) 2–11

is calculated under the assumption that protons are transferred
only through the MN state. Because WT bR and W12I bR with a fast
photocycle were used in the proton pumping assay, the pumping
activities measured are in the steady state. Thus, the small difference of 15% seems to be within experimental error. Even if protons
are pumped through the new pathway, it’s contribution is negligible because the fraction of the MX state is negligible (<0.02%)
according to the results of the numerical analysis based on the proposed model.
4. Conclusions
The aromatic interaction between W12 and F135 located vertically no farther than 5 Å into the extracellular side of the membrane and laterally at the outer rim of the bR trimer is
responsible for inter-trimer association in the hexagonal latticeof trimers. The replacement of W with F (i.e., W12F) maintains inter-trimer association. The loss of trimer–trimer interactions in
D96NW12I bR abolishes the positive and negative cooperative effects on decay kinetics as the initial ground state recovers. It also
produces 35% inactive bR molecules owing to the reduction in
the amount of all-trans retinal. Interestingly, the loss of trimer–trimer interactions reduces the rate of photo-induced conformationalchange in bR (outward displacement of the E–F loop) down to aquarter, which is very likely to be caused by the branching of thephotocycle into two pathways. Thus, trimer–trimer association
plays essential roles in providing bound retinal with an appropriate
environment to maintain its full photo-reactivity and in maintaining the natural photo-reaction pathway.
Acknowledgments
This research was supported by JST CREST (T.A.), Knowledge
Cluster/MEXT Japan (T.A.), Grants-in-Aids for Scientiﬁc Researchfrom the MEXT Japan (Nos. 15101005, T.A.; 22018012, K.I.;
19042009, T.U.; 20108014, H.K.) and a Research Fellowship of JSPS
for Young Scientists (M.S.).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jsb.2013.02.011.
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