Simulating Freely Diffusing Single-Molecule FRET Data with Consideration of Protein Conformational Dynamics.

Single-molecule Förster resonance energy transfer (smFRET) experiments have greatly contributed to the understanding of the conformational dynamics of proteins and other biomolecules. Generating high-fidelity simulated data for smFRET experiments is an important step toward developing and examining accurate and efficient smFRET data analysis techniques. Here, we use distributions of interdye distances generated using Langevin dynamics to simulate freely diffusing smFRET timestamp data for proteins and biomolecules that have conformational flexibility. We then compare analysis techniques for smFRET data to validate the new module. The Langevin dynamics is used here as an illustrative example to demonstrate how modeling conformational dynamics can be integrated with molecular diffusion and photon emission statistics, all of which are essential for realistic simulation of freely diffusing smFRET data. We also discuss different ways to generalize our approach to make the simulated data more realistic including the employment of molecular dynamics (MD) simulations that is illustrated with an example. The Langevin dynamics module provides a framework for generating timestamp data for systems with a known underlying conformational heterogeneity as a step toward the development of new analysis techniques for smFRET data dealing with flexible proteins or other biomolecular systems.