Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an analogy to an increase in the local viscosity due to the presence of the protein residues. This explanation denies the opposite effect of fluorescence quenching. Here, we present a modified perspective for understanding PIFE’s mechanism. We also describe a novel phenomenon that we name ‘protein-induced fluorescence quenching’ (PIFQ), which exhibits an opposite effect of PIFE. Our characterization of these two fluorescence modulations reveals that the initial fluorescence state of the mediator (DNA) determines whether this mediator-conjugated dye undergoes through PIFE or PIFQ, upon protein binding. This key role of the mediator DNA, which was never characterized to this day, enabled us to provide a protocol for the experimental design for obtaining, ‘on-demand’, either PIFQ or PIFE. This work will have a profound impact on the interpretation of PIFE results and will remove the arbitrary nature of its experimental design. It will also allow for proper integration of PIFE and PIFQ with existing bulk and single molecule fluorescence techniques in studying the mechanisms of nucleic acids binding proteins.
Vlad-Stefan Raducanu graduated from the Faculty of Physics, University of Bucharest, Romania. His bachelor’s degree thesis was in Theoretical Physics on the subject of “Coherent quantum states of electrons in uniform static magnetic fields”. During his bachelor’s degree studies, he came in contact with Biophysics and Biochemistry, to which he was attracted immediately. With the desire to gain more knowledge of Biology he joined KAUST for a master’s degree in Bioscience. During his master’s he joined Professor’s Samir Hamdan laboratory, in which he later continued up to date as a Ph.D. student. His focus is in understanding different processes involved in DNA Repair and Replication, through a variety of Biophysical and Biochemical methods, with an emphasis on fluorescence tools.