Direct Observation and Real-Time Tracking of an Extraordinarily Stable Folding Intermediate in Mitotic Arrest Deficient Protein 2 Folding by Single-Molecule Fluorescence Resonance Energy Transfer.

Although ensemble experiments have suggested that mitotic arrest deficient protein 2 (Mad2), a metamorphic protein, has folding intermediates, direct evidence and characterization are not available. It remains an outstanding challenge to capture the folding intermediates in real time, which is crucial to elucidate the folding mechanism, but the folding intermediates are normally unstable and only exist transiently. By combining confocal-microscopy-based and total internal reflection fluorescence (TIRF)-microscopy-based single-molecule Förster resonance energy transfer (sm-FRET) techniques, we have investigated the folding/unfolding process of Mad2 and captured its folding intermediate at the single-molecule level. This provides direct evidence for the existence of an intermediate along the folding pathway of Mad2. The folding intermediate proved to be extraordinarily stable, with an extremely long average dwell time of 2.3 s under the conditions of 3 M GdmCl at ambient temperature. The folding trajectories obtained from TIRF experiments further suggest that the intermediate is on-pathway to native Mad2.