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Dynamic visualization of protein molecules in action by highspeed AFM
Toshio Ando
Kanazawa University, Kanazawa, Ishikawa, Japan
Proteins are inherently dynamic molecules. They change the structure and interact with other
molecules dynamically, which is largely responsible for the biological functions. Therefore,
the direct real-space and real-time visualization of protein molecules at work must be a
straightforward approach to understanding the functional mechanism but the lack of suitable
techniques has precluded it. Even with the recently emerging super-resolution fluorescence
microscopes, this dream is unachievable because they are indirect imaging techniques.
Atomic force microscopy (AFM) is a versatile technique to directly image proteins in liquids
at submolecular resolution. However, its poor temporal resolution has meant an availability of
only static or slow time-lapse images of proteins. To materialize the dream, we have been
developing high-speed AFM over more than 15 years. Various devices in AFM and control
techniques were optimized or invented for high-speed scanning, and techniques were devised
to make low-invasive imaging compatible with highspeed imaging. As a result, the imaging
rate now reaches 10-30 frames/s for the scan range of 250×250 nm2 100 scan lines, and the
spatial frequency of a sample surface topography of 0.1 nm. Remarkably, even delicate
protein-protein interactions are not disturbed by the tip-sample contact. With this capacity of
high-speed AFM, some biological processes are successfully captured on video, such as
walking myosin V molecules along actin filaments, photo-activated structural changes in
bacteriorhodpsin, cooperative GroEL-GroES interactions. The molecular movies reveal their
molecular mechanisms. Thus, direct and dynamic visualization is demonstrated to be a new
approach to understanding the functional mechanisms of biological molecules.