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

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  • FUKAGAWA Akihiro
    Structural Biology Center, National Institute of Genetics Research Center for Allergy and Immunology, RIKEN
  • HIROSHIMA MichioA
    Structural Biology Center, National Institute of Genetics Research Center for Allergy and Immunology, RIKEN
  • SAKANE Isao
    Structural Biology Center, National Institute of Genetics
  • TOKUNAGA Makio
    Structural Biology Center, National Institute of Genetics Research Center for Allergy and Immunology, RIKEN Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (Sokendai) Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology

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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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