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Single-molecule experiments have changed the way we explore the physical world, yet data analysis remains time-consuming and prone to human bias. Here, we introduce Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite powered by deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, especially from single-molecule Förster Resonance Energy Transfer (smFRET) experiments. Deep-LASI automatically sorts recorded traces, determines FRET correction factors and classifies the state transitions of dynamic traces all in ~20-100 ms per trajectory. We benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data., (© 2023. Springer Nature Limited.)

The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.