Dynamics of Polymeric Protein Assemblies in Live Cells Revealed by Fluorescence Polarization Imaging of Single Molecules

Abstract

PIFE, an acronym that stands for ''Protein Induced Fluorescence Enhancement'', is a term that has been coined to describe the enhancement of fluorescence intensity that the dye Cy3 experiences in the proximity of a protein. The approach has been used to study dynamic aspects of a large number of DNAand RNA-protein interactions at the single molecule level. We and others have hypothesized that the phenomenon results from a restriction in the photoisomerization deactivation pathway of the dye. Here, we present the results of a detailed spectroscopic study that aims to characterize PIFE at the molecular level. We used time-resolved fluorescence, fluorescence anisotropy and transient spectroscopy to fully characterize the deactivation pathways of the dye on DNA when next to a protein. Our results allowed us to confirm our hypothesis that the enhancement in fluorescence correlates with a decreased yield of photoisomer.