Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 246

A Fluorescent dye proximal to phosphorylation site

Phosphorylation

P

Peptide substrate

B Binding of a phosphorecognition domain

Phosphorylation

P

Peptide substrate

C Binding to the active conformation of the kinase

Active kinase

Phosphorylation

P

Peptide substrate

Figure 6.8 Environmentally sensitive peptide-based kinase biosensors. Environmentally
sensitive peptide biosensors bear a fluorophore whose spectral properties are
directlymodified/affected by proximal phosphorylation (at 1-5 residues). Environmen-
tally sensitive peptide biosensors of kinase activity have been developed with a fluores-
cent dye coupleddirectly at or proximal to thephosphorylation sitewhose fluorescence is
enhanced (A) by phosphorylation itself, 109 (B) upon binding of a phosphopeptide recog-
nition domain such as SH2 for phosphotyrosine, 111 and (C) upon binding of the kinase
itself through an interface that does not affect its catalytic activity. 112

biosensor was engineered by a HTS approach, by derivatizing a monobody
that specifically recognizes the active conformation of Src kinases with an
environmentally sensitive merocyanine dye, the fluorescence of which is
enhanced upon target binding, yet without interfering with target binding
or phosphorylation ( Fig. 6.8C ).

3.2. Metal-ion-induced, chelation-enhanced fluorescence
Metal-ion-induced, chelation-enhanced fluorescence (CHEF) relies on
fluorophores that can coordinate metal ions (Ca 2 þ ,Mg 2 þ ,Zn 2 þ ,orlantha-
nides) and whose ability to chelate these ions is further aided by proximal
phosphorylation of a peptide substrate. Upon chelation, the electronic struc-
ture of the fluorophore is modified, and consequently its spectral properties. 137