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 (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 language | English |
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Article number | 1000 |
Journal | Nature Communications |
Volume | 13 |
Issue number | 1 |
DOIs | |
State | Published - 22 Feb 2022 |
Externally published | Yes |
Bibliographical note
Publisher Copyright:© 2022, The Author(s).
Funding
We thank Gregor Hagelücken and Martin Peter from the Institute of Structural Biology (University of Bonn, GER) for providing YopO. We would like to thank Robert Quast and Emmanuel Margeat for insightful discussions regarding the implementation of mpHMM for the analysis of 4-detector nsALEX measurements (2-color smFRET, with fluorescence anisotropies), based on their existing data. We would also like to thank Demain Lieberman for his helpful discussion regarding implementation of HMM code, and Bill Harris for his help in enabling the H2MM_C code to work on Windows and Linux. This paper was supported by the National Institutes of Health (NIH, grant R01 GM130942 to S.W. and E.L. as a subaward), the National Science Foundation (NSF, grants 1818147 and 1842951 to S.W.), the Human Frontiers Science Program (HFSP, grant RGP0061/2019 to S.W.), the Israel Science Foundation (ISF, grant 3565/20 to E.L., within the KillCorona – Curbing Coronavirus Research Program), the Milner Fund (to E.L.), and the Hebrew University of Jerusalem (start-up funds to E.L.). Work in the lab of T.C. was financed by Deutsche Forschungsgemeinschaft (SFB863, project A13 and GRK2062, project C03), an ERC Starting Grant (No. 638536 – SM-IMPORT to T.C.) and by the Center of Nanoscience Munich (CeNS). 2 2
Funders | Funder number |
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Center of Nanoscience Munich | |
National Science Foundation | 1818147 |
National Institutes of Health | |
Foundation for the National Institutes of Health | |
National Institute of General Medical Sciences | R01GM130942 |
Directorate for Biological Sciences | 1842951 |
European Commission | 638536 – SM-IMPORT |
Human Frontier Science Program | RGP0061/2019 |
Deutsche Forschungsgemeinschaft | GRK2062, SFB863 |
Hebrew University of Jerusalem | |
Israel Science Foundation | 3565/20 |