Accounting for dye diffusion and orientation when relating FRET measurements to distances: three simple computational methods

Abstract

Förster resonance energy transfer (FRET) allows in principal for the structural changes of biological systems to be revealed by monitoring distributions and distance fluctuations between parts of individual molecules. However, because flexible probes usually have to be attached to the macromolecule to conduct these experiments, they suffer from uncertainty in probe positions and orientations. One of the way to address this issue is to use molecular dynamics simulations to explicitly model the likely positions of the probes, but, this is still not widely accessible because of the large computational effort required. Here we compare three simpler methods that can potentially replace MD simulations in FRET data interpretation. In the first, the volume accessible for dye movement is calculated using a fast, geometrical algorithm. The next method, adapted from the analysis of electron paramagnetic studies, utilises a library of rotamers describing probe conformations. The last method uses preliminary MD simulations of fluorescent dyes in solution, to identify all conformational states of dyes and overlays this on the macromolecular system. A comparison of these methods in the simple system of dye-labelled polyproline, shows that in the case of lack of interaction between the dye and host, all give results comparable with MD simulations but require much less time. Differences between these three methods and their ability to compete with MD simulations in the analysis of real experiment are demonstrated and discussed using the examples of cold shock protein and leucine transporter systems.

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins

“…We generated two pairs of labeling sites for FRET donor and acceptor (939/982 and 982/1005) by mutating Thr939, Gln 982, and Asp 1005 to cysteine,r espectively (Figure 4a). [16] Thedistance distribution profiles and their variation shifts upon NuMA binding are consistent between experiment and simulation (Figure 4b,c). The donor-acceptor orientation, photophysics processing of the dye may also account for the broadening of the FRET histogram that is common in the smFRET measurement.…”

“…To compare smFRET measurements and MD simulation directly,t he FRET efficiency was converted to interdye distance,a nd the donor-acceptor distances of the MD snapshots were calculated using available volume method (see Methods in the Supporting Information). [16] Thedistance distribution profiles and their variation shifts upon NuMA binding are consistent between experiment and simulation (Figure 4b,c). In both labeling systems,the interdye distances increase upon complex formation.…”

Monomeric Huntingtin Exon 1 Has Similar Overall Structural Features for Wild-Type and Pathological Polyglutamine Lengths

“…Dye rotamers were randomly chosen from the HandyFRET rotamer libraries and dyes were attached to the protein such that the carbon–sulfur–carbon angle was approximately ideal. 61 A protein + dye conformation was accepted if no steric clashes were observed between the protein and the dye. A steric clash was defined as any protein atom being within the solvation shell of any dye atom.…”

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

“…The relative orientation of the dipole moments of the donor and acceptor molecules can significantly influence the efficiency of energy transfer [5, 41, 42, 44, 45]. This can be seen from the factor κ 2 that contributes in Eq (3).…”

“…The averaged orientation factor is often used 〈 κ 2 〉 = 2/3, [41, 42]. However, in many situations the orientation diffusion can be comparable to the time-scale of excitations or from molecular-level sterics it may not be isotropic sampling all orientations [12, 41, 44, 46]. Also, even for rapid diffusion, experimental measurements often involve a small sample of the κ 2 values which can range between 0 and 4.…”