A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Forster-Type Resonance Energy Transfer (PIFE-FRET)

作者：Lerner Eitan; Ploetz Evelyn; Hohlbein Johannes; Cordes Thorben; Weiss Shimon

来源：Journal of Physical Chemistry B, 2016, 120(26): 6401-6410.

DOI：10.1021/acs.jpcb.6b03692

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Forster-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 degrees 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.

• 出版日期2016-7-7