Transition Time Determination of Single-Molecule FRET Trajectories via Wasserstein Distance Analysis in Steady-State Variations in smFRET (WAVE).

Many biological molecules respond to external stimuli that can cause their conformational states to shift from one steady state to another. Single-molecule FRET (Fluorescence Resonance Energy Transfer) is of particular interest to not only define the steady-state conformational ensemble usually averaged out in the ensemble of molecules but also characterize the dynamics of biomolecules. To study steady-state transitions, i.e., non-equilibrium transitions, a data analysis methodology is necessary to analyze single-molecule FRET photon trajectories, which contain mixtures of contributions from two steady-state statuses and include non-equilibrium transitions. In this study, we introduce a novel methodology called WAVE (Wasserstein distance Analysis in steady-state Variations in smFRET) to detect and locate non-equilibrium transition positions in FRET trajectories. Our method first utilizes a combined STaSI-HMM (Stepwise Transitions with State Inference Hidden Markov Model) algorithm to convert the original FRET trajectories into discretized trajectories. We then apply Maximum Wasserstein Distance analysis to differentiate the FRET state compositions of the fitting trajectories before and after the non-equilibrium transition. Forward and backward algorithms, based on the Minimum Description Length (MDL) principle, are used to find the refined positions of the non-equilibrium transitions. This methodology allows us to observe changes in experimental conditions in chromophore-tagged biomolecules or vice versa.