The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.
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