On the proper retrieval of spatial information from single-molecule FRET experiments – decoupling of dynamical and photophysical effects

Physical and Analytical Chemistry Seminar

Lecturer: Prof. Eitan Lerner

20-20 Jan 2019 @ 12:30

Location: Faculty Seminar Room


Single-molecule Förster Resonance Energy Transfer (smFRET) is gaining ever-growing popularity in the study of the structure and structural dynamics of bio-macromolecules and their complexes. The structural assessment is based on the Förster relation between the efficiency of energy transfer between two dyes and the distance between them. smFRET analysis via photon distribution analysis (PDA) takes into account photon shot-noise, inter-dye distance distribution and interconversion between states to extract accurate distance information. Yet inter-dye distance fluctuations, on the timescale of the fluorescence lifetime (or shorter), can increase the observed FRET efficiency and thus give the impression of an overall decreased inter-dye distance. Additionally, photophysical processes other than FRET, such as dye photoblinking, also have to be taken into account. Although the information on diffusion-enhancement of FRET could in principle be retrieved from model fitting to fluorescence decays, in single-molecule fluorescence measurements fluorescence decays are too noisy to be accurately fitted with such complex models. Here we introduce a PDA approach dubbed Monte-Carlo diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolors photons of smFRET measurements taking into account dynamics of inter-dye distance fluctuations, multiple interconverting states and photoblinking. Using this approach, we introduce a global fitting approach for retrieving the underlying inter-dye distance distribution, decoupled from the effects of rapid inter-dye distance fluctuations and photoblinking on FRET. We show that distance interpretation of smFRET experiments of molecules as simple as doubly-labeled dsDNA (normally used for calibration of smFRET distance measurements) is nontrivial and requires decoupling the effects of rapid inter-dye distance fluctuations on FRET in order to avoid systematic biases in the estimation of the inter-dye distance distribution.