Detecting Intramolecular Dynamics and Multiple FRET States by Fluorescence Correlation Spectroscopy

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Issue Date
2010-03-06Author
Price, E. Shane
DeVore, Matthew S.
Johnson, Carey K.
Publisher
American Chemical Society
Type
Article
Article Version
Scholarly/refereed, author accepted manuscript
Rights
This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of Physical Chemistry B, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jp912125z.
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Show full item recordAbstract
Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (greenred and red-green). We applied the method to detect intramolecular dynamics in the Ca2+ signaling protein calmodulin. Dynamics were detected on the 100-μs time scale in Ca2+-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro15-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca2+-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.
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Citation
Price, E. S., DeVore, M. S., & Johnson, C. K. (2010). Detecting Intramolecular Dynamics and Multiple FRET States by Fluorescence Correlation Spectroscopy. The Journal of Physical Chemistry. B, 114(17), 5895–5902. http://doi.org/10.1021/jp912125z
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