Direct observation of multiple and stochastic transition states by a feedback-controlled single-molecule force...

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.