Simulating freely-diffusing single-molecule FRET data with consideration of protein conformational dynamics

Single-molecule Forster resonance energy transfer experiments have added a great deal to the understanding of conformational states of biologically important molecules. While great progress has been made in studying structural dynamics of biomolecular systems, much is still unknown for systems with conformational heterogeneity particularly those with high flexibility. For instance, with currently available techniques, it is difficult to work with intrinsically disordered proteins, particularly when freely diffusing smFRET experiments are used. Simu-lated smFRET data allows for the control of the underlying process that generates the data to examine if a given smFRET data analysis technique can detect these underlying differences. Here, we include a distribution of inter-dye distances generated using Langevin dynamics to simulated freely-diffusing smFRET timestamp data in order to model proteins with conformational flexibility within a given state. We compare standard analysis techniques for smFRET data to validate the new module relative to the base PyBroMo software and observe qualitative agreement in the results of standard analysis for the two timestamp generation methods. The Langevin dynamics module provides a framework for generating timestamp data with a known underlying heterogeneity of inter-dye distances that will be necessary for the development of new analysis techniques that study flexible proteins or other biomolecular systems.