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    FRET-FCS Detection of Intra-Lobe Dynamics in Calmodulin

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    Price_2011.pdf (479.6Kb)
    Issue Date
    2011-07-28
    Author
    Price, E. Shane
    Aleksiejew, Marek
    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/jp203743m.
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    Abstract
    Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca2+-signaling protein calmodulin. Intramolecular fluctuations were resolved byglobal fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius forFRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca2+ binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca2+) was nearly abolished at a high Ca2+ concentration. For the C-terminal domain the dynamic amplitude changed little with Ca2+ concentration. The Ca2+ dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca2+-binding loops.
    URI
    http://hdl.handle.net/1808/23847
    DOI
    https://doi.org/10.1021/jp203743m
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    • Chemistry Scholarly Works [506]
    Citation
    Price, E. S., Aleksiejew, M., & Johnson, C. K. (2011). FRET-FCS Detection of Intra-Lobe Dynamics in Calmodulin. The Journal of Physical Chemistry. B, 115(29), 9320–9326. http://doi.org/10.1021/jp203743m

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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