Single-molecule interaction between circularly permuted green fluorescent protein and trypsin by total internal reflection fluorescence microscopy

We have applied total internal reflection fluorescence microscopy (TIRFM) to visualize the dynamic interaction between circularly permuted green fluorescent protein (cpGFP) and trypsin at the single-molecule level. When trypsin was added, the fluorescence intensities of cpGFP mutants immobilized on the surface decreased dramatically with time. We also found that such decrease depended on the structural stability of the mutants. Furthermore, the phenomenon of ‘on-off’ blinking was observed in both the absence and presence of trypsin. Statistical analysis showed that the on-time decreased as reaction time increased.