Abstract
Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.
Highlights
Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing proteinDNA interactions
We recently used smFRET to characterize the mechanism of double flap (DF) substrate recognition by DNA replication and repair Flap Endonuclease 1 (FEN1)[25]
Known quenching mechanisms cannot explain this observed effect, since FEN1 does not contain any iron sulfur cluster and carbocyanine dyes cannot be quenched, via PET, by tryptophan residues or guanosine nucleobases[29,30]. We conclude that this phosphoramidite coupled Cy3 (pCy3) quenching is a distinct observation and refer to it as protein-induced fluorescence quenching (PIFQ), analogous to PIFE
Summary
Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing proteinDNA interactions. It is well-accepted that the isomerization in the excited state goes through a metastable 90°-twisted intermediate state during the rotation, which rapidly and non-radiatively deexcites to the ground state (Fig. 1a)[3,4,5,6,10,11] Within this paradigm, PIFE has been explained by an analogy to an increase in the local viscosity due to the presence of the protein residues decreasing the rate of photoisomerization from trans S1* to twisted S1* (Fig. 1a). Existing models rely on the idea that proteins influence the rate of photoisomerization from trans S1* to twisted S1* through the concepts of steric hindrance/restriction[17] and specific contact with certain residues[3,4] These models predict only fluorescence enhancement and deny the existence of an opposite phenomenon given that the presence of the protein can only increase the local viscosity. If the dye is in contact with the same protein residues, these models assume that the change in fluorescence will not depend on the initial fluorescence of the DNA-
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