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Real-Time Atomic Force Microscopy

Structural Changes in Bacteriorhodopsin in
Response to Alternate Illumination
Observed by High-Speed Atomic ForceMicroscopy

Blue light, green light: High-speed atomic
force microscopy visualized light-induced
structural changes of the D96N bacteriorhodopsin (bR) mutant under alternate
two-color illumination. With green light,
each bR molecule is displaced outward
from the trimer center. This activatedstructure is driven back to the groundstate by the subsequent blue-light illumination (see picture).

M. Shibata, T. Uchihashi, H. Yamashita,
H. Kandori, T. Ando*4410 – 4413

Keywords: chromophores ·electron microscopy · membrane proteins ·nanotechnology · protein structures

2011 – 50/19

 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Communications
DOI: 10.1002/anie.201007544

Real-Time Atomic Force Microscopy

Structural Changes in Bacteriorhodopsin in Response to Alternate
Illumination Observed by High-Speed Atomic Force Microscopy**
Mikihiro Shibata, Takayuki Uchihashi, Hayato Yamashita, Hideki Kandori, and Toshio Ando*
The membrane protein bacteriorhodopsin (bR), found in
Halobacterium salinarum, functions as a light-driven proton
pump transferring protons across the membrane from thecytoplasmic side to the extracellular side.[1] bR is comprised ofseven transmembrane a helices (named A–G) surrounding
the retinal chromophore covalently bound to Lys216 througha protonated Schiff base.[2] bR assembles into trimers, which
are packed into two-dimensional hexagonal lattices, the socalled purple membrane.[3] Upon absorption of light, photoisomerization from the all-trans to the 13-cis conformation ofretinal takes place, with subsequent primary proton transferfrom the Schiff base to Asp85, which triggers a cascade ofchanges in bR structure. A series of intermediates designatedJ, K, L, M, N, and O have been defined by spectroscopy, andM (M410, having a light absorbance peak at 410 nm) is the onlyintermediate containing a deprotonated Schiff base.[1]The light-induced conformational changes in bR havebeen studied by various methods.[4–8] Particularly importantare crystallographic structures in the frozen activated state of
the wild type (WT) and of bR mutants, which were solved at
atomic resolution.[6] The common understanding regarding
the bR structure during the photocycle is that the proton
channel on the cytoplasmic surface is opened by the tilting of
helix F away from the protein center.[6] This alteration isfollowed by rearrangement of the interhelix E–F loop, thus
resulting in large-scale conformational changes in the M andN intermediates.[5, 7, 8]
Atomic force microscopy (AFM), which can visualize
nanometer-scale objects in various environments,[9] has also
been used for structural studies of bR under aqueousconditions, and high-resolution images that allow the iden-[*] Dr. M. Shibata, Prof. Dr. T. Uchihashi, Dr. H. Yamashita,
Prof. Dr. T. Ando
Department of Physics, Kanazawa University
Kakuma-machi, Kanazawa (Japan)
Fax: (+ 81) 76-264-5739
E-mail: tando@kenroku.kanazawa-u.ac.jp
Homepage: http://www.s.kanazawa-u.ac.jp/phys/biophys/index.htm
Prof. Dr. H. Kandori
Department of Frontier Materials, Nagoya Institute of Technology
Showa-ku, Nagoya (Japan)[**] This work was supported by JST CREST (T.A.), Knowledge Cluster/
MEXT Japan (T.A.), Grants-in-Aids for Scientific Research from JSPS
(Basic research S, No. 20221006, T.A.) and by MEXT Japan (No.
19042009, T.U.; No. 20108014, H.K.) and Research Fellowships of
JSPS for Young Scientists (M.S). We thank Prof. Leonid S. Brown forcomments on the draft.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201007544.

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tification of individual interhelix loops are obtained.[10, 11] To
date, conventional AFM has provided structural information
not only of bR but also of other membrane proteins.[10–12]
However, conventional AFM can only show static or slow
time-lapse images of biomolecules because of its poor
temporal resolution. Over the last decade, however, thescan speed of AFM has been increased by various efforts.[13–15]
Recent advances in fast scanning techniques of AFM havedemonstrated that it is possible to observe dynamic behaviorof single protein molecules in action.[16]Using high-speed atomic force microscopy (HS-AFM),
we recently succeeded in the real-space and real-timeobservation of light-induced conformational changes of bR
under physiological conditions.[17] The photocycle of the wild
type at neutral pH values proceeds very quickly (ca. 10 ms),and hence, the conformational changes cannot be clearly
imaged even by our HS-AFM. To slow down the photocycle,
we used the D96N bR mutant, which has a longer photocycle(ca. 10 s at pH 7) but still retains a proton pumping ability.[18]Reversible dynamic structural changes in bR in response to
green light appeared in the molecular movies.[17]It is known that the all-trans to 13-cis isomerization ofretinal can be reversed by subsequent illumination with lightof a different wavelength.[19] After M410 is formed upon green
illumination, it can be driven back to the ground state (bR570)
by blue light. However, the spectroscopically detected
reversal does not necessarily indicate the reversal of theentire protein structure. Namely, it is not certain to datewhether the protein structure is tightly coupled to theconformation of retinal. Herein, we address this issue by
directly visualizing D96N bR under alternate illumination
with green (l = 532 nm) and blue (l = 408 nm) light using HSAFM.
Figure 1 presents AFM images of the D96N bR mutant at
the cytoplasmic surface captured at 1 frame s 1 (see Movies S1 and S2 in the Supporting Information). Under thegreen light, a part of each bR molecule is displaced counterclockwise and outward from the trimer center (compare
images at 15 and 28 s in Figure 1), as we reported.[17] As aresult, three nearest-neighbor bR monomers, each of which
belongs to a different adjacent trimer, are brought into
contact with each other. Figure 2 shows the displacement of
the centers of mass as a function of time for three different bRmonomers (M1–M3 in Figure 2). Note that each “center ofmass” was calculated from the height distribution at a surface
region of each bR monomer in the AFM image (see theSupporting Information for details). Under these illumination
conditions, once bR molecules change their conformation bygreen light, the conformation of most bR molecules appears
to remain in the activated state even after the light is turned 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Angew. Chem. Int. Ed. 2011, 50, 4410 –4413

Figure 1. HS-AFM images of the D96N bR mutant at the cytoplasmic
surface. 1 frame s 1, 200  200 pixels. The membranes were adsorbed
onto a mica surface in 10 mm phosphate (pH 8) and 300 mm KCl. A
bR trimer is highlighted by the white triangles. The white arrows
indicate the direction of the conformational change in bR (15 s). The
green bars indicate application of 532 nm green light (27 and 28 s).
The insets in the images at 15 and 28 s are averaged images of a bR
trimer captured in the dark and under green light, respectively.

Figure 2. Displacement of centers of mass for three D96N bR molecules measured as a function of time at pH 8 on the cytoplasmic side.
The average displacement induced by light is (0.80  0.13) nm(mean  standard deviation, s.d.). The green bars show the periods ofgreen illumination.

off. The molecules behave in this way because the lifetime of
the activated state of D96N bR at pH 8 is longer than the
duration of the on–off cycle of illumination.
The protruding parts of bR around helices E and F
displace outward from the trimer center under illumination.
In addition, a previous static AFM imaging study of bR
assigned the prominent protrusion in the AFM topographs ofbR to the cytoplasmic E–F loop.[10] Thus, we conclude that theobserved conformational change of bR originates from thedisplacement of the E–F loop. There have been a number ofreports on the displacement of helices on the cytoplasmic sideof bR detected by X-ray and electron diffraction techniques.[6]However, the diffraction techniques are generally unable todetermine the displacement of loop regions because of thedisordered structures. In contrast, electron paramagnetic
Angew. Chem. Int. Ed. 2011, 50, 4410 –4413

resonance (EPR) spectroscopy can detect the E–F loop
displacement as a change in the distance between a pair ofnitroxide spin labels attached to the E–F loop and to anotherlocus on the cytoplasmic surface. Thorgeirsson et al. reported
that upon photoactivation, the distance between 103C (in the
C–D loop) and 163C (in the E–F loop) of the A103C/M163C
double mutant and the distance between 35C (in the A–Bloop) and 163C of the S35C/M163C double mutant increaseby 0.65 and 0.4 nm, respectively.[5] From various interspindistance measurements, Xiao et al. suggested that the E–F
loop is displaced counterclockwise and outward from theprotein center.[7] These studies suggest that the E–F loop is
displaced by a larger distance than the helices move. Our
observation of the counterclockwise rotation and displacement of the EF loop by approximately 0.8 nm is consistent
with these EPR studies.
Figure 3 shows images of the cytoplasmic surface underalternate illumination with green and blue light. In contrast to
the case of only green light, the analysis of the center of massof each molecule clearly shows that bR molecules undergoalternate conformational changes, which are mostly synchron-

Figure 3. HS-AFM images of D96N bR mutant at the cytoplasmic
surface illuminated by green and blue light. The green and blue bars
indicate application of 532 nm green light (23 and 29 s) and 408 nm
blue light (36 and 37 s). Blue arrows highlight an instance of bR
activation by blue light.

ized with the alternate application of green and blue light(Figure 4; see also Movies S3 and S4 in the Supporting
Information). The blue-light-induced conformational changeback to the ground state is caused by the photo-back-reaction,
not by the normal turnover of the photocycle. Because bR in
the ground state also absorbs blue light, deactivated bR issometimes reactivated under the blue light (blue arrows at 36
and 37 s in Figure 3, Movie S3 in the Supporting Information,and blue arrows in Figure 4). These observations clearly showthat the conformation of bR is tightly coupled to theconformation of retinal.

 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Communications
molecules is a powerful new approach to study conformational changes in proteins induced by external stimuli.

Experimental Section

Figure 4. Displacement of centers of mass for three D96N bR molecules measured at pH 8 on the cytoplasmic side. The green and blue
bars show periods of green and blue light application, respectively.
Blue arrows indicate bR activation by blue light.

Sample preparation: Purple membranes containing the D96N bR
mutant were isolated from Halobacterium salinarum as described.[17, 20] The samples were suspended in a solution containing
10 mm phosphate buffer (pH 8) and 300 mm KCl.
HS-AFM: The experimental conditions of the high-speed AFM
measurements were similar to those reported previously.[17] The AFM
images were obtained in the tapping mode for the single-layered
purple membranes on a mica surface under solution at room
temperature. To detect the cantilever deflection, we used an opticalbeam deflection detector equipped with an infrared laser (980 nm).
The laser beam was focused onto a small cantilever using a  50
objective lens. The cantilevers (Olympus) are 6–7 mm long, 2 mm wide,and 90 nm thick with a spring constant of 0.1–0.2 N m 1. Their

Next, we examined whether the activatedstate of bR observed by AFM contains not
only M410 but also other intermediates thatare spectroscopically different from M410. To
address this issue, the photocycle was initiated by brief green-light illumination, andthen blue light was briefly applied withdifferent delay times. Figure 5 a shows the
decay of the activated state after the green
light was applied for 3 s. Monomers whose
centers of mass are displaced outward by
approximately 0.8 nm are counted as being in
the activated state. After the green light is Figure 5. Decay of the activated state of D96N bR detected by HS-AFM at pH 8. Greenswitched off, bR molecules thermally return and blue regions indicate application of green and blue light, respectively. Illumination
to the ground state through the photocycle periods of both green and blue light are 3 s. a) Decay through the normal course of the(Figure 5 a), with a time constant of about photocycle with a time constant of (39  0.27) s (mean  s.d., the total number of analyzed
40 s. In contrast, Figure 5 b shows the decay bR molecules n = 354). b) Decay induced by blue light (n = 389).
of the activated state induced by blue lightapplied at a delay time of 30 s after turning
resonant frequency and quality factor in an aqueous solution are
off the green light. Although a few bR molecules areapproximately 1 MHz and 2, respectively. An amorphous carbon tip
deactivated and then reactivated during the blue-light illumiwas grown on the original tip by electron beam deposition. The tip
length was adjusted to approximately 1 mm, and the tip apex was
nation, these molecules are not counted in this analysis.
sharpened by plasma etching under argon gas (ca. 4 nm radius). The
Before the blue light is turned on, the fraction of activatedbR sample was irradiated with green (532 nm) or blue (408 nm) laser
molecules gradually decreases in the normal course of thelight (both ca. 0.5 mW) through the  50 objective lens.
photocycle, but it is abruptly decreased nearly to zero after
the brief blue-light illumination (Figure 5 b and Figure S1 in
Received: December 1, 2010
the Supporting Information). The time constant for the decay
Published online: April 6, 2011
during the blue-light illumination is about 1 s, independent of
the delay time between the two illumination periods
Keywords: chromophores · electron microscopy ·(Table S1 in the Supporting Information). Thus, we conclude
membrane proteins · nanotechnology · protein structuresthat the conformation of D96N bR in the activated state
observed by AFM solely corresponds to the M410 intermediate, which efficiently absorbs blue light.
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