Multi-parameter photon-by-photon hidden Markov modeling

Paul David Harris, Alessandra Narducci, Christian Gebhardt, Thorben Cordes, Shimon Weiss, Eitan Lerner

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (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 cause artifacts. 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.

Original languageEnglish
Article number1000
JournalNature Communications
Issue number1
StatePublished - 22 Feb 2022
Externally publishedYes

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