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.

Fluorescence resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

“…In the preceding section we have shown that the PIFE-ruler has a clear signature in ALEX experiments that renders it a useful tool for mechanistic biomolecular studies This data suggests that the dynamic range of the PIFE-FRET assay regarding distance R1 (the range between S for Cy3 and for Cy3B, for a given E value) is optimal at low FRET efficiencies 52 . Accurate FRET values 49 are required to obtain distances on the FRET axis, i.e., distance R2.…”

“…Ultimately, we think that an optimized To finally relate the distance dependencies of the PIFE-ruler present here to published smPIFE work, the fold increase in Cy3 fluorescence QY due to the PIFE effect is needed, which is only indirectly available in ALEX. In a forthcoming paper 52 , we provide the needed photophysical framework for ALEX-based PIFE-FRET to demonstrate its ability to obtain fully quantitate experimental results from ALEX that are directly comparable with published smPIFE studies. The nature of the model described in ref.…”

“…have to be considered since these alter the observed "apparent" PIFE effect. The effect of steric hindrance serves as a lower boundary to the PIFE effect 52 and Cy3 at some bases (3 and 5 bp from interaction with BamHI) can have a larger PIFE effects than expected just from steric hindrance, which can only be explained by additional specific interactions.…”

“…Restricting the rotational freedom of the fluorophore by applying steric hindrance/restriction 73,74 , and also possibly specific interactions with select protein residues, the interacting protein causes a delay in photoisomerization to dark excitedstate isomers 52,58 , which, in turn, increases fluorescence intensity without chromatic changes ( Fig. S1).…”

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

“…This range was extended to shorter distances by exploiting the phenomenon of self-quenching of identical fluorophores (8), quenching by radical (9), quenching through the electron transfer with a tryptophan or tyrosine residue (10,11), or the proteininduced fluorescence enhancement (PIFE) (12). FRET was also combined with PIFE to gain a concomitant information about molecular interactions on different distance scales (13). There were also attempts of nanoscopic ruling at distances exceeding the upper FRET limit by using the interaction of two metallic nanoparticles (14) or a metallic particle and a fluorophore (15).…”