Format

Send to

Choose Destination
See comment in PubMed Commons below
Biophys J. 2012 Mar 7;102(5):1163-73. doi: 10.1016/j.bpj.2011.11.4025. Epub 2012 Mar 6.

Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis.

Author information

1
Department of Chemistry, Ben-Gurion University of the Negev, Be'er-Sheva, Israel.

Abstract

Among the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided.

PMID:
22404939
PMCID:
PMC3296060
DOI:
10.1016/j.bpj.2011.11.4025
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science Icon for PubMed Central
    Loading ...
    Support Center