Multi-parameter photon-by-photon hidden Markov modeling.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 35194038.
- Also identified by DOI 10.1038/s41467-022-28632-x and PMC identifier 8863987.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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<sup>2</sup>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<sup>2</sup>MM (mpH<sup>2</sup>MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH<sup>2</sup>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<sup>2</sup>MM facilitates the identification and quantification of biomolecular sub-populations and their origin.
Medical subject headings
- Fluorescence Resonance Energy Transfer
- Photons