Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3

Protein-induced fluorescence enhancement (PIFE) is a term used to describe the increase in fluorescence intensity observed when a protein binds to a nucleic acid in the proximity of a fluorescent probe. PIFE using the single-molecule dye Cy3 is gaining popularity as an approach to investigate the dynamics of proteins that interact with nucleic acids. In this work, we used complexes of DNA and Klenow fragment and a combination of time-resolved fluorescence and transient spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein (PIFE). By monitoring the formation of the cis isomer directly, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.