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
