title: A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET).
abstract: Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles , 607 Charles E. Young Drive East, Los Angeles, California 90095-1569, United States.
authors.forename: Eitan
authors.initials: E
authors.lastname: Lerner
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Ploetz
authors.affiliation: Laboratory of Biophysics, Wageningen University and Research , Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.|Microspectroscopy Centre, Wageningen University and Research , Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hohlbein
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Thorben
authors.initials: T
authors.lastname: Cordes
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles , 607 Charles E. Young Drive East, Los Angeles, California 90095-1569, United States.
authors.forename: Shimon
authors.initials: S
authors.lastname: Weiss
mesh_terms: D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D007536:Isomerism; D007700:Kinetics; D008962:Models, Theoretical; D055668:Photochemical Processes
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; C089187:cyanine dye 3; D004247:DNA; D004262:DNA Restriction Enzymes