Analytical Sciences

Direct Observation of Multiple and Stochastic Transition States by a Feedback-controlled Single-molecule Force Measurement

Akihiro FUKAGAWA,*¹,*² Michio HIROSHIMA,*¹,*² Isao SAKANE,*¹ and Makio TOKUNAGA*¹,*²,*³,*⁴

*1 Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
*2 Research Center for Allergy and Immunology, RIKEN, Yokohama, Kanagawa 230-0045, Japan
*3 Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (Sokendai), Mishima, Shizuoka 411-8540, Japan
*4 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori, Yokohama, Kanagawa 226-8501, Japan

To overcome the ensemble-averaging barrier, single-molecule experiments have been performed, but energy landscapes comprising multiple intermediates have not yet been defined. We performed mechanical unfolding of staphylococcal nuclease using intermolecular force microscopy, modified AFM with high resolution and feedback control of the positioning. The force dropped vertically just after its peak, and multiple transition states were detected as force peaks. The multiple and stochastic intermediates found in the present study provide new important information on protein folding.

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