Journal of Structural Biology 167 (2009) 153–158

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

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic
force microscopy
Hayato Yamashita a,*, Kislon Voïtchovsky b,1, Takayuki Uchihashi a,c,
Sonia Antoranz Contera b, John F. Ryan b, Toshio Ando a,ca

Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
Physics Department, University of Oxford, Parks Road, OX1 3PU, Oxford, UK
c
CREST/JST, Sanban-cho, Chiyoda-ku, Tokyo, Japanba r t i c l e

i n f oArticle history:
Received 21 January 2009
Accepted 28 April 2009
Available online 3 May 2009
Keywords:
High-speed atomic force microscopy
Bacteriorhodopsin
Two-dimensional crystal
Dynamic equilibrium
Trimer
Molecular interactiona b s t r a c t
We have used high-speed atomic force microscopy to study the dynamics of bacteriorhodopsin (bR) molecules at the free interface of the crystalline phase that occurs naturally in purple membrane. Our results
reveal temporal ﬂuctuations at the crystal edges arising from the association and dissociation of bR molecules, most predominantly pre-formed trimers. Analysis of the dissociation kinetics yields an estimate of
the inter–trimer single-bond energy of 0.9 kcal/mol. Rotational motion of individual bound trimers
indicates that the inter–trimer bond involves W10–W12 tryptophan residues.
Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction
The current view of cell membrane structure derives from theﬂuid mosaic model in which proteins are considered to diffuse
freely within a ﬂuid lipid bilayer (Singer and Nicolson, 1972). The
ﬁrst direct evidence for protein diffusion within cell membranes
was provided by hybrid cell experiments (Frye and Edidin, 1970).
Since then, various techniques including ﬂuorescence recovery after
photobleaching microscopy (Swaminathan et al., 1997) and single
particle tracking microscopy (Kusumi et al., 1993) have provided amore detailed understanding of the mobile nature of proteins in biological membranes. In particular, it has been shown that proteins in
native membranes may not diffuse freely but are in fact conﬁned to
speciﬁc domains. Cells use several conﬁning mechanisms such as
anchoring to the cytoskeleton through hetero-bifunctional proteins(Byers and Branton, 1985), diffusion barriers formed by the accumulation of proteins anchored to cytoskeleton meshes (Halenda et al.,
1987; Nakada et al., 2003), or self-assembly into large two-dimensional (2D) crystalline patches, as in the case of bacteriorhodopsin(bR), a light-driven proton pump found in the so-called purple
membranes (PM) of Halobacterium salinarum (Blaurock and Stoeckenius, 1971). Despite these advances, an understanding of the* Corresponding author. Fax: +81 762 64 5739.
E-mail address: yhayato@stu.kanazawa-u.ac.jp (H. Yamashita).
1
Present address: Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Mass. Ave., Cambridge, USA.
1047-8477/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.jsb.2009.04.011

membrane dynamics at the nanoscale remains a major challenge
due primarily to the lack of measurement techniques allowing
simultaneous spatial and temporal observation of single molecules
within native membranes.
In this paper, we introduce a new technique for observing membrane protein dynamics, high-speed atomic force microscopy (HSAFM) (Ando et al., 2001, 2008), which provides both high spatial(1 nm) and temporal (40 ms/image) resolution of single molecules in their native environment. We have used this techniqueto study the dynamics of bR crystal formation at the edges of native
PM. PM is composed of bR trimers which are arranged in a hexagonal lattice (Henderson et al., 1990). We directly imaged the dynamic events occurring at the interface between crystalline anddisordered areas of PM. Fitting our results with a simple model,
we were able to estimate the energy of the trimer–trimer interaction and the degree of oligomerization of bR in the non-crystal region. Our results also highlight the role played by certain residuesof bR (W10–W12) in the interactions between trimers.
2. Materials and methods
2.1. Sample preparation
PM from the NRL strain of Halobacterium salinarum was isolated
following standard procedures (Oesterhelt and Stoeckenius, 1974).
The purest fraction obtained by sucrose density gradient centrifugation was frozen and stored at 193 K. After thawing and then

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H. Yamashita et al. / Journal of Structural Biology 167 (2009) 153–158

removing sucrose by sedimentation, the pellet was suspended in asolution (Buffer-A) containing 10 mM Tris–HCl (pH 8.0) and
300 mM KCl. The sample (1.7 mg/ml) was kept at 277 K before
AFM experiments. For AFM observation, the sample was diluted
to 0.1 mg/ml in Buffer-A.
2.2. HS-AFM observation
The laboratory-built tapping mode HS-AFM apparatus usedhere is an extensively improved version of the AFM previously reported (Ando et al., 2001). The cantilever deﬂection sensor was
specially optimized for bR observations and equipped with a
980 nm infrared laser. The laser beam was focused onto a smallcantilever using a 50 objective lens (Olympus). The cantilever(Olympus) has a resonant frequency of 1 MHz in water and aspring constant of 0.1–0.2 N/m. An amorphous carbon tip wasgrown on the original cantilever tip by electron beam deposition.
The tip length was adjusted to about 1 lm and the tip apex was
sharpened by plasma etching (4 nm in radius) (Ando et al.,2008). A sample stage made of quartz glass was placed on the zscanner and a 1.5 mm diameter mica disk was glued onto the sample stage. A 2 ll sample droplet was then deposited on the freshly
cleaved mica surface and incubated for 3 min. All HS-AFM measurements were performed under solution (Buffer-A) at room temperature. Both the membrane and the atomically ﬂat mica
substrate possess a strong hydration shell which prevents directbinding of PM to the mica, and the adsorption occurs throughnon-speciﬁc electrostatic forces. A water-layer of thickness
1 nm separates the membrane from the mica surface allowing

motion of membrane proteins within the membrane (Kim et al.,
2001; Sackmann, 1996).
Control experiments with simple supported lipid bilayers ensured that the effective experimental temperature of the sample
was lower than 310 K, judging from the gel phase transition of
the lipids (not shown). To estimate the inter–trimer association energy in the photo-activated state, a green laser (532 nm, 20 lW)
was directed through the objective lens onto the sample. This
low-power irradiation is sufﬁcient for exciting all bR moleculesin the observed area but does not damage the sample.
3. Results and discussion
3.1. High-resolution images of 2D crystal of bacteriorhodopsin in PM
PM exhibits ﬂat, round-shape patches typically 1–5 lm indiameter. In the patch shown in Fig. 1A, a 2D crystal lattice of bR
is formed over the inner region (surrounded by the dotted line),
whereas in the peripheral outer region, there is no crystalline order. Fig. 1B shows a high magniﬁcation image of the inner regionof the cytoplasmic leaﬂet captured at 1 frame/s. The distinctionof PM cytoplasmic and extracellular sides was made using thehigh-resolution AFM image as previously reported (Müller et al.,
1996; Voitchovsky et al., 2006). The hexagonal lattice of bR trimers
is clearly visible, demonstrating the ability of our HS-AFM apparatus to visualize individual bR molecules even at high imaging rate.
Two higher magniﬁcation images of an edge region of PM acquired
successively at 1 frame/s (with an interframe time of a few ms) arepresented in Fig. 1C and D, respectively. They both exhibit a dis-Fig. 1. AFM images of PM adsorbed onto a mica surface. (A) Low magniﬁcation image. PM patches consist of a crystal area (encircled with the dotted line) and a non-crystal
area (the periphery of the crystal area). (B) High magniﬁcation image of the crystal area showing the packing arrangement of bR molecules at the membrane cytoplasmic
surface. The bR trimers (encircled with the triangles) are arranged in a hexagonal lattice with unit cell dimension of 62 Å. (C) and (D) are successive AFM images of an edge
region of PM. Scale bars: (A) 80 nm, (B) 10 nm, and (C and D) 20 nm. Vertical brightness range: (A) 13 nm, (B) 0.8 nm, (C) 7.3 nm, (D) 7.5 nm.

H. Yamashita et al. / Journal of Structural Biology 167 (2009) 153–158

tinct border between the crystal and non-crystalline area. The
shape of the border differs between the two images (arrows), indicating that the crystal is in dynamic equilibrium with bR molecules
located in the non-crystalline area. In fact, ‘‘noise” spikes were frequently observed in the non-crystalline area, most likely produced
by diffusing bR molecules moving too fast to be clearly resolved at
1 frame/s.
3.2. Association and dissociation of bR molecules at crystal edges
To investigate the details of the dynamic structural changes
occurring at the crystal edge, a time sequence of higher magniﬁcation images of the boundary region was acquired (see Fig. 2 andSupplementary movie 1). The darker regions in the images correspond to non-crystalline areas. The bR trimers in the crystal are
indicated by the thinlined triangles. At 0.6 s (Fig. 2A), two bR trimers (red triangles) have newly bound to the crystal edge. At
2.1 s (Fig. 2A), two bR trimers (white triangles) have dissociatedand another trimer (red triangle) has bound to the crystal edge.
One of the two dissociated trimers remained in the crystal areafor 0.9 s. Not only bR trimers but also bR dimers and monomers
were observed to bind to and dissociate from the crystal edge. A
bR dimer (red rectangle in Fig. 2B) stayed bound to the edge for0.5 s, whereas a bR monomer (red circle in Fig. 2C) remainedbound to the edge for 0.4 s. These residence times for monomers
and dimers are shorter than those of the trimers. Table 1 summarizes 239 observed binding events, classiﬁed according to theiroligomerization states. The binding of trimeric bR occurred predominantly (82%), whereas binding of dimeric bR (6.7%) was only
about half that of monomeric bR (11.3%).

155

3.3. Pivotal motion of weakly bound bR trimer
On rare occasions, we observed large crevasses in the crystal as
exempliﬁed in Fig. 3A (area surrounded by the dotted line). In thecrevasse, the two-dimensional lattice structure is not present, andconsequently bR trimers are loosely packed and exhibit appreciable motion, which is restricted to some extent by the interaction
with the surrounding bR molecules. At the interface between thedense crystal area and the non-crystalline area, pivotal motion ofbR trimers was often observed. As shown by the AFM images taken
at 25 frames/s (Fig. 3B), the bR trimer (red triangle) rotated counterclockwise around the inter–trimer bond (arrow, compare theimages at 0 s and 0.24 s) and subsequently oscillated around thesame position for more than 2 s. At 2.88 s, the trimer rotated backto its initial position (see Supplementary movie 2). This pivotal
motion was repeatedly observed. Although we noted similar motion at the distinct interface between the crystalline and non-crystalline areas like the one shown in Fig. 2, the pivotal motion was
too fast to be clearly imaged.
We interpret this pivotal motion as a direct effect of the inter–
trimer bonds. Static images of PM with molecular or submolecularresolution reveal the parts of a bR molecule which lie in close proximity with the neighboring trimers (Fig. 1B). However, such images
do not necessarily show the actual location of the inter–trimer
bonds since such proximity may occur passively and non-speciﬁcally. On the other hand, HS-AFM movies can localize the main inter–trimer bond from the trimer pivot point. Neutron diffraction
studies and structural comparison with halorhodopsin (hR) indicate that a tryptophan residue (W12) participates in lattice formation (Oesterhelt, 1995; Weik et al., 1998). In addition, it has beenFig. 2. Time-lapse high-magniﬁcation AFM images of the borders between the crystal and non-crystal areas. The bR molecules encircled by the red dotted lines indicate
newly bound bR trimer (A), dimer (B) and monomer (C). The white triangles indicate the previously bound trimers (at 2.1 s in (A)). Scale bars: (A–C) 10 nm. Imaging rate: (A)
3.3 frames/s, (B and C) 10 frames/s. Vertical brightness range: (A) 3.8 nm, (B) 2.6 nm, (C) 2.8 nm.

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H. Yamashita et al. / Journal of Structural Biology 167 (2009) 153–158

Table 1
Observed binding events classiﬁed according to the oligomerization states of the
newly bound bR.

Number of events
Fraction (%)

Trimer

Dimer

Monomer

196
82.0

16
6.7

27
11.3

shown that the double mutation (W12I/W80I) leads to collapse of
the 2D bR crystal and monomerization of bR trimers (Sapra et al.,2006). Furthermore, force spectroscopy experiments have highlighted the role of W 10–W 12 in anchoring bR into the PM latticethrough steric-speciﬁc interactions (Voitchovsky et al., 2007).
Fig. 3C shows a schematic reconstruction of Fig. 3B using the pdb
structure of bR trimers (Essen et al., 1998) with the positions ofFig. 3. (A) AFM image of bR crystal with a crevasse. (B) Time-lapse high magniﬁcation AFM images on the border between the crystalline and non-ordered areas. The bR
trimer highlighted by the red triangle rotated around a pivot (white arrow) formed at the crystal edge (compare the images taken at 0 s and at 0.24 s) and then oscillated for2 s. At 2.88 s, the trimer rotated back to its initial position. (C) Schematic model for the observed AFM images. The positions of W12 in the bR model are indicated by whitespheres (PDB 1BRR). The white arrows point at the interaction bonds. The dotted white circle indicates the pivot region for the trimer. Scale bars: (A) 20 nm, (B) 10 nm.
Imaging rate: (A) 2.5 frames/s, (B) 25 frames/s. Vertical brightness range: (A) 2.2 nm, (B) 1.4 nm.

H. Yamashita et al. / Journal of Structural Biology 167 (2009) 153–158

W12 highlighted (white spheres). When comparing the AFM
images with the corresponding model, the trimer appears to pop
out of the crystal due to breaking of bonds localized near W12 residues (indicated by the arrows in Fig. 3C), subsequently it rotates
through another W12 residue (circled by the dotted line) and is ﬁnally re-trapped at the crystal edge.
3.4. Quantitative analysis of inter–trimer interaction energy
In order to estimate the inter–trimer interaction energy, we
analyzed the residence time of newly bound bR trimers at the crystal edge and its dependence on the number of interaction sites. In
addition, we examined the effect of bR photo-activation on the residence time since the protein photo-isomerization can, in principle,
affect the membrane dynamics at larger scale (Manneville et al.,1999). For our analysis, we assumed that within the two-dimensional bR crystal, a trimer can interact with the surrounding trimers through six sites, each based on a W12 residue as indicatedby the dotted lines in ‘VI’ of Fig. 4A. Following the same model,
the number of interaction sites at the crystal edge is reduced,
depending on the binding position, as indicated by ‘I’, ‘II’ and ‘III’in Fig. 4A. Successive AFM images as exempliﬁed in Fig. 2 showed
many binding and dissociation events in which bR trimers boundA

to different sites at the border between the crystal and non-crystal
areas. These events can be classiﬁed into types ‘I’, ‘II’ and ‘III’
depending on the number of interaction sites involved. Type IIbinding events are predominant (74%), whereas type I (6%)and type III (20%) events are less frequent. The lifetime of thetype I bonds was too short to obtain clear images of the corresponding event, preventing reliable statistics.
Fig. 4B shows a histogram of the lifetime of type II bonds which
was measured using AFM images taken at 10 frames/s (tip velocity,
75 lm/s). This histogram could be well ﬁtted by a single exponential (correlation coefﬁcient, r = 0.9), from which the average lifetime s2 was estimated to be 0.19 ± 0.01 s. To ensure that theobserved dissociation events are not signiﬁcantly affected by the
AFM tip during the scanning, we examined the dependence of
the average lifetime on the tip velocity when a constant scanning
force is maintained. The inset in Fig. 4B shows the average lifetime
as a function of the tip velocity and indicates that the average lifetime is about 0.17 ± 0.06 s, irrespective of the tip velocity. Thus, weconclude that the tip-sample interaction does not signiﬁcantly affect the natural association and dissociation kinetics of the bR trimer. Fig. 4C shows a histogram of the type III bond lifetime, fromwhich the average lifetime s3 was estimated to be 0.85 ± 0.08 s.
The longer lifetime of type III bonds compared with type II obviously arises from a relationship of E3 < E2 < 0, where E2 and E3 are
the association energies responsible for type II and type III interactions, respectively, (Yamamoto et al., 2008). The average lifetime
ratio, s2/s3, is given bys2 =s3 ¼ exp½ðE3  E2 Þ=K B T

Number of Events

B 40

1.0

30

0.5

20

0.0

10
0

60
80
100
Tip Velocity [ µm/s]=0.19 sec
0

1
2
Bond Lifetime [s]C 30
Number of Events

Bond II

3

Bond III25
20=0.85 sec

15
10
5
0

0

1

2
3
4
5
Bond Lifetime [s]67Fig. 4. (A) Schematic representation of the binding of a trimer at the crystal edge (I–III) and in the crystal interior (VI). Roman numerals indicate the number ofinteraction bonds (dotted lines) involving the W12 residue. (B and C) Histogram
showing the type II and III binding events vs. lifetime. These histograms were ﬁtted
with a single-exponential function (red line). The inset in (B) shows the average
lifetime as a function of the tip velocity. Error bars indicate the standard deviation
for the nonlinear least-square curve ﬁtting.

157

ð1Þ

where kB is Boltzmann’s constant and T is the temperature in Kelvin.
Because the type II interaction contains two elementary bonds,
whereas the type III interaction contains three (see Fig. 4A), the energy difference E3  E2 corresponds to the association energy of thesingle elementary bond. From the ratio s2/s3 = 0.22 and Eq. (1), thiselementary association energy is estimated to be about 1.5 kBT,
which corresponds to –0.9 kcal/mol at 300 K.
A previous study of differential scanning calorimetry on the
2D bR crystal showed two calorimetric transitions (Jackson andSturtevant, 1978): a reversible transition at 70–80 °C and an irreversible transition at 100 °C. The reversible transition wasinterpreted as melting of the lattice structure, which was later
conﬁrmed by X-ray diffraction studies of hydrated PM (Koltover
et al., 1999). A low degree of order is indeed retained at temperatures slightly above the melting temperature, which likely corresponds to bR trimers. The energy required for melting thelattice structure is estimated to be 6–7 kcal/mol (Jackson andSturtevant, 1978). This value approximately coincides with
6  0.9 kcal/mol estimated by our AFM study for the association energy in the full interaction of a trimer with the surrounding lattice (type VI).
In order to assess the possible effect of photo-induced structuralchanges of bR on the PM lattice dynamics, we measured s2 and s3
for illuminated PM. However, we did not observe any appreciable
difference with the corresponding lifetimes measured for dark
adapted membrane. Our observation contrasts with the results of
Kahya et al. who reported a decreased lateral mobility for photoactivated bR molecules due to formation of clusters comprising
two or three trimers (Kahya et al., 2002). Their results, however, relied on ﬂuorescence correlation spectroscopy and freeze-fracture
electron microscopy of bR reconstituted in model membranes(giant unilamellar vesicles). We believe this discrepancy can be explained by differences in the constituent lipids of the native andmodel membranes. PM native lipids are indeed known to be essential for bR crystallization (Sternberg et al., 1993) and dynamics(Hendler and Bose, 2003).

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4. Conclusion
We have succeeded in measuring association-dissociation
dynamics of bR at the edges of a native PM crystal at the single
molecule level using HS-AFM. The results provide a direct observation of the mechanisms of PM lattice co-translational assembly andstrongly support the hypothesis that bR trimers are preformed before being assembled into the crystal (Krebs and Isenbarger, 2000).
Our AFM movies also highlight the presence of a pivot point for therotational motion of bR trimers bound to the PM edge region, demonstrating the presence of a speciﬁc inter–trimer bond near theW12 residue. Consistently, the average residence times of bR trimers bound to the crystal edge depended on the number of bonds
formed, allowing us to estimate the association energy of the single
bond to be 0.9 kcal/mol, in good agreement with differential
scanning calorimetry measurements. Thus, our approach provides
a good estimate of the inter–trimer association energy. As demonstrated here with PM, the unique ability of HS-AFM to provide
simultaneous high spatial and temporal resolution opens new avenues for studying the dynamics of native biological systems.
Acknowledgments
We acknowledge Dr. Daisuke Yamamoto for valuable discussions. This work was supported by the JST CREST project, the JST
project of Development of Systems and Technology for Advanced
Measurement and Analysis, and the Strategic International Cooperative Program of the JST with a Grant-in-Aid for Scientiﬁc Research
on Priority Areas, a Grant-in-Aid for Basic Research (S) from the
JSPS Japan, and a Grant-in-Aid for JSPS Fellows (to H.Y.). This work
was also supported by the Bionantechnology IRC and a BBSRC JPA.
K.V. acknowledges funding from the Lord Florey fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jsb.2009.04.011.
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