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    Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Perdios, L. and Lowe, Alan R. and Saladino, G. and Bunney, T.D. and Thiyagarajan, N. and Alexandrov, Y. and Dunsby, C. and French, P.M.W. and Chin, J.W. and Gervasio, F.L. and Tate, E.W. and Katan, M. (2017) Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET. Scientific Reports 7 , p. 39841. ISSN 2045-2322.

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    Abstract

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

    Metadata

    Item Type: Article
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Depositing User: Administrator
    Date Deposited: 13 Jun 2017 12:17
    Last Modified: 27 Jul 2019 22:33
    URI: http://eprints.bbk.ac.uk/id/eprint/18757

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