Measuring distance changes with one dye (Hwang et al., 2011). (a) Calibration of PIFE (protein induced fluorescence enhancement) effect as a function of distance obtained from exemplary proteins: restriction enzyme, BamHI, and a motor protein RIG-I, and comparison to FRET vs distance. (b) As RIG-I translocates on RNA-DNA duplex, fluorescence signal of a fluorophore attached to one end of the duplex changes, decreasing as the protein moves away from the fluorophore. (c) A single molecule fluorescence intensity time trace showing repetitive translocation of RIG-I observed via PIFE. (d) Time traces averaged over about 50 translocation cycles over duplexes of 20, 30, and 40 base pairs in length.