Förster resonance energy transfer: Role of diffusion of fluorophore orientation and separation in observed shifts of FRET efficiency

PLoS One. 2017 May 19;12(5):e0177122. doi: 10.1371/journal.pone.0177122. eCollection 2017.

Abstract

Förster resonance energy transfer (FRET) is a widely used single-molecule technique for measuring nanoscale distances from changes in the non-radiative transfer of energy between donor and acceptor fluorophores. For macromolecules and complexes this observed transfer efficiency is used to infer changes in molecular conformation under differing experimental conditions. However, sometimes shifts are observed in the FRET efficiency even when there is strong experimental evidence that the molecular conformational state is unchanged. We investigate ways in which such discrepancies can arise from kinetic effects. We show that significant shifts can arise from the interplay between excitation kinetics, orientation diffusion of fluorophores, separation diffusion of fluorophores, and non-emitting quenching.

MeSH terms

  • Diffusion
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry*
  • Kinetics

Substances

  • Fluorescent Dyes

Grants and funding

This work was supported by National Science Foundation Career Grant 0956210, National Science Foundation: Division of Mathematical Sciences 1616353 (www.nsf.gov), and Department of Energy Advanced Scientific Computing Research CM4 DESC0009254 (http://science.energy.gov/ascr/).