Katan, M. and Perdios, P. and Lowe, Alan R. and Saladino, G. and Bunney, D.T. and Thiyagarajan, N. and Alexandrov, Y. (2016) Conformational transition and dynamics of 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 Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Depositing User: | Administrator |
| Date Deposited: | 09 Jan 2017 09:03 |
| Last Modified: | 02 Aug 2023 17:29 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/17625 |
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