SUPPLEMENTARY INFORMATION
Supplementary information for:doi: 10.1038/nnano.2010.7

High-speed atomic force microscopy reveals dynamic
molecular processes in photo-activated bacteriorhodopsin

Mikihiro Shibata1, Hayato Yamashita1, Takayuki Uchihashi1,2, Hideki Kandori3 andToshio Ando1,2*

1Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan

2

CREST/JST, Tokyo 102-0075, Japan3

Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555,

Japan*e-mail: tando@kenroku.kanazawa-u.ac.jp

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supplementary information

doi: 10.1038/nnano.2010.7

M1

Distance from initial position (nm)

M2
M3
M4
M5

M6
M7

M8M9
M101.5 nm

0306090120150Time (s)Supplementary Figure 1 І Displacement of the mass-center position from its initialposition plotted as a function of time for 10 D96N bR monomers at the cytoplasmic
surface (Supplementary Movie 1). The green regions indicate the illumination periods.
The average displacement of the mass-center position is 0.69 ± 0.15 nm. Although theindividual molecules show different lifetimes of the activated state, the initial bR
recovers within 1 s. In addition, even under illumination (within green regions), themass-center position often moves back to the initial position; some monomers repeat thephoto-induced conformational changes under illumination (especially M5 and M6).

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supplementary information

doi: 10.1038/nnano.2010.7

a5 nm

0s

1.0 s

2.0 sb3.0 sc
M15 nmDistance from initial position (nm)

M2

M3EDCBAF GM4M5M6M7M8M9M101.5 nm

0306090120150Time (s)Supplementary Figure 2 І High-speed AFM images of the extracellular surface ofD96N under dark or illuminated conditions. a, Successive AFM images of D96N bRadsorbed onto a mica surface in 10 mM Tris-HCl (pH 7) and 300 mM KCl. Frame rate:
1 fps; pixel size: 200×200 pixels (Supplementary Movie 5). A bR trimer is highlighted
by the white triangles. The first two frames were captured in the dark condition,
whereas the last two frames were captured under illumination of 532-nm green lightwith 0.5 μW (the green bars indicate illumination). b, Displacement of the mass-center
position from its initial position plotted as a function of time for 10 bR monomers(Supplementary Movie 5). The green regions indicate the illumination periods. c,
Enlarged AFM image of D96N at the extracellular surface in the unphotolyzed state. AbR trimer is highlighted by the white triangle, and the position of the α-helicalextracellular ends (indicated by A–G) is derived from an atomic model of theunphotolyzed state. Traces of the mass-centers are superimposed on the AFM image(red, blue, and black marks).
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supplementary information
M5 1

Distance from initial position (nm)M11

1.5 nmdoi: 10.1038/nnano.2010.7

M 12

M5 2

M1 3

M5 3

M6 1

M2 1

M2 2

M6 2

M2 3

M6 3

M3 1

M7 1

M3 2

M7 2

M3 3

M7 3

M4 1

M8 1

M4 2

M8 2

M4 3

M8 3

060120180240300360420480060120180240300360420480

Time (s)Time (s)Supplementary Figure 3 І Displacement of mass-center position for D96N at thecytoplasmic surface under different light intensities. The figures show displacementof the mass-center position as a function of time measured for 24 bR monomers withineight different trefoils under different light intensities at pH 7 (Supplementary Movie 4).
Here, “trefoil” means a triad of nearest neighbor bR molecules, each of which belongs
to a different adjacent trimer. Mn1-Mn3 (n=1-8) indicate monomers within the n-thtrefoil. During imaging, the light intensity was gradually increased in eight steps. The
green regions correspond to the illumination periods, and their shade level indicates therelative intensity of the illumination. With increasing light intensity, the frequency ofconformational changes is increased except for M41 and M81 monomers. In contrast,
M23, M43, M61, M73, and M83 monomers change their conformations even under4

weaker illumination.

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supplementary information

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Supplementary Movie 1 І High-speed AFM movie of the cytoplasmic surface of
D96N at pH 7 during dark-illumination cycles. The purple membranes containing theD96N bR mutant were adsorbed onto a mica surface in 10 mM Tris-HCl (pH 7) and 300
mM KCl. Frame rate: 1 fps; pixel size: 150×150 pixels. The green bars shown in theAFM movie indicate green-light illumination. A bR trimer is highlighted by the white
triangle at the first frame. This movie is played at ×10 speed.
Supplementary Movie 2 І High-speed AFM movie of the cytoplasmic surface ofWT at pH 7 during dark-illumination cycles. The purple membranes containing WTbR were adsorbed onto a mica surface in 10 mM Tris-HCl (pH 7) and 300 mM KCl.
Frame rate: 1 fps; pixel size: 150×150 pixels. The green bars shown in the AFM movie
indicate light illumination. A bR trimer is highlighted by the white triangle at the firstframe. This movie is played at ×10 speed.
Supplementary Movie 3 І High-speed AFM movie of the cytoplasmic surface of
WT at pH 10 during dark-illumination cycles. The purple membranes containing WTbR were adsorbed onto a mica surface in 10 mM Tris-HCl (pH 10) and 300 mM KCl.
Frame rate: 1 fps; pixel size: 150×150 pixels. The green bar shown in the AFM movie
indicates light illumination. A bR trimer is highlighted by the white triangle at the firstframe. This movie is played at ×10 speed.
Supplementary Movie 4 І High-speed AFM movie of the cytoplasmic surface of
D96N at pH 7 with increasing light intensity. The purple membranes containing
D96N bR were adsorbed onto a mica surface in 10 mM Tris-HCl (pH 7) and 300 mM
KCl. Frame rate: 1 fps; pixel size: 200×200 pixels. While imaging, the light intensity
was gradually increased in eight steps. The green bars indicate the illumination periods,

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doi: 10.1038/nnano.2010.7

and their shade level indicates the relative light intensity. A bR trimer is highlighted bythe white triangle at the first frame. This movie is played at ×10 speed.
Supplementary Movie 5 І High-speed AFM movie of the extracellular surface of
D96N at pH 7 during dark-illumination cycles. The purple membranes containing
D96N bR were adsorbed onto a mica surface in 10 mM Tris-HCl (pH 7) and 300 mM
KCl. Frame rate: 1 fps; pixel size: 200×200 pixels. The green bars shown in the AFM
movie indicate light illumination. A bR trimer is highlighted by the white triangle at thefirst frame. This movie is played at ×10 speed.

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