Multi-parameter photon-by-photon hidden Markov modeling

Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (H 2 MM) 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 cause artifacts. Here, we introduce multi-parameter H 2 MM (mpH 2 MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH 2 MM 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 mpH 2 MM facilitates the identification and quantification of biomolecular sub-populations and their origin. In this work, the authors demonstrate the application of multi-parameter photon-by-photon hidden Markov modeling (mpH 2 MM) on alternating laser excitation (ALEX)-based smFRET measurements. The utility of mpH 2 MM in identifying and quantifying dynamic biomolecular sub-populations is demonstrated in three different systems.