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bioRxiv · 10.1101/2021.04.08.439035

Multi-parameter photon-by-photon hidden Markov modeling

Harris, P. D.·Weiss, S.·Lerner, E.
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Abstract

Single-molecule Forster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacro-molecules. Photon-by-photon hidden Markov modelling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may introduce artefacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.

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Harris, P. D., Weiss, S., Lerner, E.. 2021-04-10. Multi-parameter photon-by-photon hidden Markov modeling. https://doi.org/10.1101/2021.04.08.439035

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