Madoka Yamaguchi, Masahiro Nakano, Ryosuke Senga, Hiroyuki Maruyama, Shige H. Yoshimura, and Yoshikazu Nakayama
Single-molecule force measurement, Carbon nanotube, Sprcific molecular interaction, Decoration of AFM cantilever tips
Single-molecule measurement of biological intermolecular interaction force provides a powerful means for the fundamental investigation of life sciences. We developed a technology for site-selective trapping of a protein to a tip of carbon nanotube (CNT) probes for use with atomic force microscopy. A labeled importin α was covalently trapped site-selectively via its azido group at the CNT probe tip. Using this technique, CNT probe tips trapping importin α at its importin β binding (IBB) region or other region were prepared to measure the interaction force against randomly oriented importin β attached on a substrate. The importin α, where the IBB region was free, interacted specifically and nonspecifically, but importin α with an attached IBB region was only able to interact nonspecifically. The importin β which had already bound to importin α was also examined to provide only nonspecific interaction. Consequently, we obtained the specific interaction force between individual importin α and importin β and reported that the force is related logarithmically to the loading rate of force. This result enables determination of the width of the potential barrier for unfolding and the intrinsic unfolding rate constant in the absence of force between importin α and importin β.
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