Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 252

significant enhancement of the fluorescence of the environmentally sensitive
probe ( Fig. 6.11B ). This strategy has proved extremely successful in generating
a PKA biosensor bearing a pyrene probe, using Rose Bengal as a quencher and
a 14-3-3pi as a phosphoserine-binding domain, which displays 64-fold in-
crease in fluorescence, 129 and more recently, another PKA biosensor bearing
a Coumarin probe and Acid Green as a quencher, which displays up to 150-
fold increase in fluorescence, was used to monitor PKA activity in mitochon-
dria. 130 Despite its apparent potency, the complexity of this approach makes it
difficult to apply in living cells and in vivo .
3.3.3 Quenching through aggregation
Avariant of these strategies was devised by Sun et al. based on an “auto-assembly”
or “micelle” approach to quench fluorescence of peptide kinase biosensors
forPKA,PKC,p38,MAPKAPK2,Akt,Erk1,andSrc. 131 This involves the
design of an amphiphilic peptide biosensor labeled with fluorescein, composed
of a substrate domain with a phosphorylatable residue and of an N-terminal car-
bon tail that promotes auto-assembly or aggregation of the peptides, thereby
quenching their fluorescence. Phosphorylation of the peptide biosensor disrupts
the micelle structure, thereby leading to fluorescence ( Fig. 6.11C ).
3.4. Ratiometric strategies
3.4.1 Intramolecular ratiometric strategy — Single-probe biosensor
Two different ratiometric strategies have been developed. The first consists
of a single probe that is sensitive to environmental changes that will affect its
spectral properties sufficiently to allow for ratiometric quantification.
A metal-ion, anion-sensitive ratiometric peptide kinase biosensor has been
developed to probe PKA activity, consisting of a Cd(II)-cylcen appended
aminocoumarin coupled to a substrate peptide for PKA. Upon phosphory-
lation by PKA, the metal-complex moiety binds to a phosphorylated
residue, which in turn displaces the coumarin fluorophore, resulting
in ratiometric change of the probe's excitation spectrum 132 ( Fig. 6.12A ).
A different strategy is based on the incorporation of two fluorophores within
the same biosensor, the first displaying sensitivity to target recognition and
the second being inert. This concept was employed to generate the Src
merobody biosensor, through incorporation of a merocyanine dye onto
the fibronectin biosensor scaffold, as well as the mCerulean autofluorescent
protein for ratio imaging in vivo 112 ( Fig. 6.12B ).