The effect of Brownian motion of fluorescent probes on measuring nanoscale distances by Förster resonance energy transfer

Abstract

Förster resonance energy transfer (FRET) is a powerful optical technique to determine intra-molecular distances. However, the dye rotational motion and the linker flexibility complicate the relationship between the measured energy transfer efficiency and the distance between the anchoring points of the dyes. In this study, we present a simple model that describes the linker and dye dynamics as diffusion on a sphere. Single-pair energy transfer was treated in the weak excitation limit, photon statistics and scaffold flexibility were ignored, and different time-averaging regimes were considered. Despite the approximations, our model provides new insights for experimental designs and results interpretation in single-molecule FRET. Monte Carlo simulations produced distributions of the inter-dye distance, the dipole orientation factor, κ(2), and the transfer efficiency, E, which were in perfect agreement with independently derived theoretical functions. Contrary to common perceptions, our data show that longer linkers will actually restrict the motion of dye dipoles and hence worsen the isotropic 2∕3 approximation of κ(2). It is also found that the thermal motions of the dye-linker system cause fast and large efficiency fluctuations, as shown by the simulated FRET time-trajectories binned on a microsecond time scale. A fundamental resolution limit of single-molecule FRET measurements emerges around 1-10 μs, which should be considered for the interpretation of data recorded on such fast time scales.

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

“…We are particularly interested in the case when the measured molecule’s conformational state involves a sampling over an ensemble of different configurations. In this case, the donor and acceptor could undergo significant translational diffusion over the donor lifetime [51, 52]. For instance, for a disordered protein or a polymer subjected to different solvation conditions FRET could be used to get an indication of the radius of gyration [35, 53–55].…”

The Internet of Molecular Things Based on FRET

“…From this expression, we can infer that when the transition dipoles are orthogonal to each other, κ 2 will be 0, and FRET does not occur. For the configurations where fluorophores are free to rotate in all there axes, or a FRET pair is self-assembled on a backbone structure without any control over transition dipoles, κ 2 would be a random variable with the following probability distribution [53]: In many experimental works studying an ensemble of FRET pairs, mean value of κ 2 , i.e., 2/3 is used with the assumption of free rotation [1].…”

Excitable Oil Droplets ‐ FRET Across a Liquid‐Liquid Phase Boundary

“…Such systems are extremely sensitive to changes in distance between donor and acceptor and the effect decreases as the sixth power of the distance separating the two. In common FRET cascades the donor and acceptor molecules are located in one phase so that the distance over which FRET occurs is of the dimensions of the Förster radius . When FRET occurs, the fluorescence intensity of the acceptor increases while a decrease in donor fluorescence intensity and fluorescence lifetime takes place .…”