Ensemble FRET simulations of conformational distance distributions in N–C swapped fluorescent protein fusions

This repository contains the Wolfram Mathematica code used for the ensemble FRET simulations described in the manuscript, “Topology-dependent FRET efficiency in living cells via N-C swapping of fluorescent protein fusions.” A related version of the manuscript is available as a preprint on bioRxiv (Tanida et al., 2026; DOI: 10.64898/2026.06.03.729767) and is currently under consideration at Scientific Reports.

The code models chromophore distances as Gaussian distributions to examine how conformational fluctuations influence ensemble FRET efficiencies. It was used to generate the pseudocolor contour maps relating effective mean chromophore distance (μeff), conformational fluctuation (σ), and theoretical ensemble FRET efficiency, as well as to calculate the effective mean chromophore distances required to reproduce the experimentally observed FRET efficiencies in living cells for the donor-acceptor (DA) and acceptor-donor (AD) configurations.

The simulations were performed for the N–C-swapped fluorescent protein fusion constructs EGFP-mCherry (DA), mCherry-EGFP (AD), EYFP-mCherry (DA), and mCherry-EYFP (AD), and were used to evaluate the relationship between conformational fluctuations and DA/AD asymmetry in effective mean chromophore distance (Figs. 7 and 8 of the manuscript).

The code was developed and executed using Wolfram Mathematica 15.0 (Wolfram Research, Champaign, IL, USA).