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


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.

SUBMITTER: Perdios L 

PROVIDER: S-EPMC5206623 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Perdios Louis L   Lowe Alan R AR   Saladino Giorgio G   Bunney Tom D TD   Thiyagarajan Nethaji N   Alexandrov Yuriy Y   Dunsby Christopher C   French Paul M W PM   Chin Jason W JW   Gervasio Francesco Luigi FL   Tate Edward W EW   Katan Matilda M  

Scientific reports 20170103


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 pr  ...[more]

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