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Reviews in Fluorescence 2017 pp 303–317Cite as

Resolving Internal and Global Dynamics of Proteins by Combination of Time-Resolved Fluorescence Anisotropy and Fluorescence Correlation Spectroscopy

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Abstract

Time-resolved fluorescence anisotropy (TRFA) is a well-established method for studying molecular dynamics that occur on a timescale from picoseconds to tens of nanoseconds. This method can be used to investigate ligand-dependent changes in the local motions of regions of interest (for example loop dynamics) in proteins and protein assemblies. TRFA data are generally fitted to models based on sums of exponentials, and cross-correlation between fitting parameters can significantly affect the accuracy and precision of the recovered parameters that are associated with local and global motions. Here, fluorescence correlation spectroscopy (FCS) data is used to obtain an independent measure of the whole molecule motion and calculate the mean rotational correlation time, which is then used to reduce cross-correlation with the TRFA parameters associated with the local motions.

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Acknowledgments

The authors are grateful for support from National Institutes of Health Grant R01GM105993 (S.R. Sprang). Fluorescence measurements and analyses were performed in the BioSpectroscopy Core Research Laboratory at the University of Montana, which is operated with support from NIH Centers of Biomedical Research Excellence (CoBRE) Award P20GM103546 to the Center for Biomolecular Structure and Dynamics and from the Vice President of Research and Creative Scholarship at the University of Montana. The authors also acknowledge helpful discussions with colleagues Stephen R. Sprang and Celestine Thomas as well as technical assistance from Michelle Terwilliger.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Montana, Missoula, MT, USA

    Labe A. Black & J. B. Alexander Ross

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  1. Labe A. Black
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  2. J. B. Alexander Ross
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Correspondence to J. B. Alexander Ross .

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Editors and Affiliations

  1. Institute of Fluorescence, University of Maryland Baltimore County, Baltimore, MD, USA

    Chris D. Geddes

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Black, L.A., Alexander Ross, J.B. (2018). Resolving Internal and Global Dynamics of Proteins by Combination of Time-Resolved Fluorescence Anisotropy and Fluorescence Correlation Spectroscopy. In: Geddes, C. (eds) Reviews in Fluorescence 2017. Reviews in Fluorescence. Springer, Cham. https://doi.org/10.1007/978-3-030-01569-5_12

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