Indexed on: 09 Feb '17Published on: 09 Feb '17Published in: Journal of Physical Chemistry B
Förster resonance energy transfer (FRET)-based single molecule techniques have revolutionized our understanding of conformational dynamics in biomolecular systems. Recently a new single molecule technique based on protein-induced fluorescence enhancement (PIFE) has aided studies where minimal (<3nm) displacements occur. Concerns have been raised regarding whether donor fluorophore intensity (and correspondingly emission quantum yield) fluctuations, intrinsic to PIFE methods, may adversely affect FRET studies when retrieving the donor-acceptor dye distance. Here we initially show through revisions of Förster's original equation that distances may be calculated in FRET experiments regardless of protein-induced intensity (and emission quantum yield) fluctuations occurring in the donor fluorophore. We additionally demonstrate via an analysis of recorded emission intensity and competing decay pathways that PIFE and FRET methods may be conveniently combined providing parallel complementary information in a single experiment. Single molecule studies conducted with Cy3- and ATTO647N-labelled RNA structures and the HCV NS5B polymerase protein undergoing sliding dynamics along the RNA backbone, provide a case study to validate the results. The analysis behind the proposed method enables for PIFE and FRET changes to be disentangled when both FRET and PIFE fluctuate over time following protein arrival and e.g. sliding. A new method Intensity-FRET (I-FRET) is thus proposed to monitor conformational changes spanning from angstroms to nanometers.