BPS2026 – Investigating heterogeneous folding transitions paths of fast-folding protein using three-color single-molecule FRET in zero-mode waveguides

Although protein folding dynamics has been successfully described by effective one-dimensional diffusion along a reaction coordinate, the underlying dynamical pathways can be heterogeneous. Transition paths (TPs) are a collection of heterogeneous pathways that encodes mechanistic information about transitions connecting stable conformational states. Distribution of the TPs describes the heterogeneity and the folding mechanism. In this study, we focus on characterizing the folding TPs of a fast-folding three-helix bundle, α3D, which have been predicted to be heterogenous by all-atom MD simulations. In principle, it can have multiple TPs of helix (H) formation, such as H1 - H2 - H3, H2 - H1 - H3, etc . Experimentally, the average transition path time of α3D is short (∼12 μs) determined by two-color single-molecule FRET. This presents experimental challenges of characterizing the TP distribution, including limited fluorescence brightness to resolve short-lived transition paths and difficulty of measuring correlated conformational motions through FRET efficiencies across multiple dye pairs. In this study, we report our progress on probing transition paths of α3D using three-color single-molecule FRET spectroscopy in zero-mode waveguides. In this experiment, α3D was site-specifically labeled with three different fluorophores to enable simultaneous tracking of all inter-helix distances, and FRET efficiencies across all three dye-pairs were obtained by pulsed-interleaved excitation. The fluorescence brightnesses were enhanced by the zero-mode waveguides, allowing us to monitor fast μs motions of helical formation in α3D. The distribution of folding TPs will be discussed.