Computational refinement of spectroscopic FRET measurements

Abstract

This article supplies raw data related to a research article entitled "Joint refinement of FRET measurements using spectroscopic and computational tools" (Kyrychenko et al., 2017) [1], in which we demonstrate the use of molecular dynamics simulations to estimate FRET orientational factors in a benchmark donor-linker-acceptor system of enhanced cyan (ECFP) and enhanced yellow (EYFP) fluorescent proteins. This can improve the recalculation of donor-acceptor distance information from single-molecule FRET measurements.

Keywords: ECFP; EYFP; FRET; Fluorescent protein; Single molecule, Molecular dynamics simulations.

Fig. 1

Scheme of an ECFP-donor and EYFP-acceptor FRET pair linked by a flexible peptide bridge (GGSGGS)₃ referred to as ECFP-l₃-EYFP.

Fig. 2

(A) The series of the four different starting configurations of ECFP-l₃-EYFP (runs 1–4) were run in parallel to achieve adequate convergence statistics. (Left) MD snapshots of the initial conformations of ECFP-l₃-EYFP. (Right) MD snapshots of final equilibrium conformation of ECFP-l₃-EYFP, taken at the end of the MD sampling.

Fig. 3

Parallel MD sampling of ECFP-l₃-EYFP. The series of the four different starting configurations of ECFP-l₃-EYFP (run 1–4) were run in parallel to achieve adequate convergence statistics.

Fig. 4

MD simulations of ECFP-to-EYFP distance. (A) Snapshot of ECFP-l₃-EYFP in a MD box in the presence of 0.1 M buffering ions Na⁺ (cyan balls) and Cl⁻ (mauve balls). (B) Comparison of ECFP-to-EYFP distance distributions calculated in the absence (blue) and in the presence of 0.1 M NaCl (magenta).

Fig. 5

(A) The transition dipole moments of the ECFP donor and EYFP acceptor chromophores are shown as vectors µ_D and µ_A, respectively. The angles Θ_D and Θ_A define the dipole orientations with respect to the connecting unit distance vector R_DA, whereas φ is the angle between the two planes. (B) The definition of the orientation factor κ².

Fig. 6

Comparison of the MD-simulated distribution of <κ²> (cyan) with the theoretical curve for an ideal unrestricted isotropic dynamic orientation of dipoles <κ²>=2/3 (blue).

Fig. 7

(A) Comparison of the experimentally observed sm-FRET efficiency (grey bars) and MD-reconstructed distribution of FRET probability. (B) Comparison of ECFP-to-EYFP distance distribution histograms calculated by the direct MD sampling (blue) and reconstructed from the MD-estimated E_DA, under the standard simplifying assumption of unique R₀=48 Å (green) (Raw data associated with this Figure are provided in Supplementary files).

Fig. 8

Comparison of True and Apparent distance distributions in ECFP-l₃-EYFP model FRET system. True Donor-to-Acceptor distance distribution (red) is calculated directly from MD trace without any assumptions. Apparent Donor-to-Acceptor distance distributions are calculated from either experimental sm-FRET data (black) or MD-generated FRET data, assuming an average orientation factor <κ²>=0.69 (and subsequently unique value of R₀=48 Å) for the entire population of conformations. (Raw data associated with this Figure are provided in Supplementary files).