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Figure 1.
(a) Structural model of a protein kinase of interest with the
DFG motif (orange) and activation loop (red) highlighted. A
cysteine was mutated into the activation loop of cSrc for
subsequent labeling with the environmentally sensitive
fluorophore acrylodan to generate a sensitive DFG-out
fluorescence-based binding assay. The DFG-out conformation is
stabilized by the binding of allosteric type III inhibitors
(blue surface representation) or type II inhibitors that lock
the kinase in the inactive state. The binding of ATP or type I
inhibitors (yellow surface representation) stabilizes the active
DFG-in conformation. Both conformations are in equilibrium and
result from structural changes in the activation loop and the
DFG motif. (b) Examples of type I, type II and type III kinase
inhibitors and scaffolds. (c) In the absence of ligand,
acrylodan-labeled cSrc shows two emission maxima at 475 nm and
505 nm. Type I ligands induce a robust loss of fluorescence
intensity (represented by red arrows) at 475 nm, resulting in a
red shift in the emission maxima to  510
nm (right panel). Type II and III inhibitors stabilize the
inactive kinase conformation and elicit a different response in
which the emissions at 475 nm and 505 nm are equally reduced
(left panel). The emission signal at 445 nm is less sensitive to
ligand binding and serves as an internal reference point,
allowing for more stable ratiometric fluorescence measurements
and K[d] determinations.
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