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

  • Labe A. Black
  • J. B. Alexander RossEmail author
Part of the Reviews in Fluorescence book series (RFLU)


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.


Protein dynamics Time-resolved fluorescence anisotropy Fluorescence correlation spectroscopy Diffusion Global analysis 



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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© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of MontanaMissoulaUSA

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