Instrumentation of the High-speed AFM andObservation of Protein Dynamics
Toshio Ando
Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
Core Research for Evolutional Science and Technology (CREST) of the Japan Science andTechnology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
E-mail: tando@kenroku.kanazawa-u.ac.jp
WWW: http://www.s.kanazawa-u.ac.jp/phys/biophys/index.htm
X-ray crystallography and NMR have been successful in determining the protein 3D structure
at atomic level. However, the obtainable structure is a static one averaged over manymolecules and hence cannot reveal how protein molecules behave dynamically when they arefunctioning in solution. Currently-prospering single molecule analysis by fluorescencemicroscopy can detect dynamic behavior of protein at work but the spatial resolution is nothigh enough to visualize protein structure. Atomic force microscopy (AFM) does not possess
spatial resolution as high as x-ray crystallography and NMR but very unique in its ability tovisualize individual protein molecules in solution at (sub) nanometer resolution. However, its
imaging rate is too low to capture dynamically moving molecules because of the slow scan
speed due to the slow mechanical response of the cantilever and scanner. In addition, the tipsample interaction force is large, which disturbs weak protein-protein interaction andsometimes leads to destruction of protein. In order to afford AFM an ability to trace moving
protein molecules without disturbing their physiological function, we have been developing
various devices over the past decade; for example, small cantilevers1 with a high resonancefrequency (0.6-1.2 MHz in water) and a small spring constant (~0.2 N/m), an opticaldeflection detection system1,2 compatible with small cantilevers, a high-speed scanner3 that
does not vibrate when operated in z-direction at 150 kHz, a dynamic PID controller4 that does
not lower the feedback bandwidth even when the amplitude set point is adjusted very close to
the cantilever’s free oscillation amplitude, a high-speed phase detector5 that allows
simultaneous capturing of topography and phase-contrast images. By these devises, it has
recently become possible to image at (near) video rate, without disturbing protein’sphysiological functions6,7. For example, hand-over-hand movement of myosin V along actin
filaments is clearly imaged. The negatively cooperative binding events between Gro-ES andthe two rings of GroEL are successfully captured. These demonstrate that high-speed AFM istruly useful for studying protein’s dynamic action and will surely open a new way ofelucidating the mechanisms of protein functions.
1. T. Ando, N. Kodera, E. Takai, D. Maruyama, K. Saito and A. Toda, Proc. Natl. Acad. Sci. USA
98: 12468-12472 (2001).
2. T. Ando, N. Kodera, E. Takai, D. Maruyama, K. Saito and A. Toda, Jpn. J. Appl. Phys.41: 48514856 (2002).
3. N. Kodera, H. Yamashita and T. Ando, Rev. Sci. Instrum. 76: 053708 (5pages) (2005).
4. N. Kodera, M. Sakashita, and T. Ando, Rev. Sci. Instrum. 77(8): 083704 (7 pages) (2006).
5. T. Uchihashi, H. Yamashita, and T. Ando, Appl. Phys. Lett. 89: 213112 (2006).
6. T. Ando, T. Uchihashi, N. Kodera, A. Miyagi, R. Nakakita, H. Yamashita, and M. Sakashita, Jpn.
J. Appl. Phys. 45(3B): 1897-1903 (2006).
7. T. Ando, T. Uchihashi, N. Kodera, A. Miyagi, R. Nakakita, H. Yamashita, & K. Matada, e-J. Surf.
Sci. Nanotech. 3: 384-392 (2005).