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.
Department of Chemistry and Biochemistry and the Biodesign Institute, Arizona State University, P.O. Box 875601, Tempe, Arizona 85287, United States.
Elana M S
EM
Stennett
Department of Chemistry and Biochemistry and the Biodesign Institute, Arizona State University, P.O. Box 875601, Tempe, Arizona 85287, United States.
Monika A
MA
Ciuba
Department of Chemistry and Biochemistry and the Biodesign Institute, Arizona State University, P.O. Box 875601, Tempe, Arizona 85287, United States.
Su
S
Lin
Department of Chemistry and Biochemistry and the Biodesign Institute, Arizona State University, P.O. Box 875601, Tempe, Arizona 85287, United States.
Marcia
M
Levitus
D002232:Carbocyanines / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D007536:Isomerism; D008027:Light; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins; C089187:cyanine dye 3; D004247:DNA; D004256:DNA Polymerase I
FRET; cyanine; isomerization; photophysics; protein−DNA interactions; single-molecule fluorescence