Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 249

developed to probe p38 a kinase in cell lysates in a selective fashion, by
incorporating an MEF2A docking peptide and p38 a phosphorylation site
bearing a CSox amino acid two residues before the phosphorylation site
into the same scaffold 123 ( Fig. 6.9D ).
Although this strategy is extremely powerful, it should be kept in mind
that the spatial constraint and proximity required between the phosphory-
lation site and the position of the chelating fluorophore may be a limitation
for recognition by certain kinases.
3.2.3 Zn 2 þ -chelating biosensors
Zn 2 þ chelators have been developed based on binuclear Zn(II)-dipicolylamine
(DPA) complexes spanned by a fluorescent module (stilbene, anthracene, phe-
nyl, or biphenyl) that is sensitive to the recognition of phosphate by the Zn 2 þ
moiety 139 ( Fig. 6.10A ). Based on these Zn 2 þ chelators, a hybrid biosensor was
designed for enhanced phosphopeptide recognition based on the
phosphopeptide-bindingWWdomain of Pin1 coupled with the Zn 2 þ chemo-
sensor 124 ( Fig. 6.10B ). This combinatorial strategy was successfully employed to
monitor phosphorylation of a pSer-CTD peptide by CDK9/cyclin T1.
3.2.4 Lanthanide-chelating biosensors
Yet another class of environmentally sensitive probes worth mentioning
report on lanthanide ions (Eu 3 þ ,Tb 3 þ ), and constitute extremely attractive
tools to develop biosensors, given their photophysical features. 140 Lantha-
nide biosensors display poor affinity for lanthanides (such as Eu 3 þ or
Tb 3 þ ) in their unphosphorylated state and are essentially nonfluorescent,
compared to their phosphorylated counterparts whose affinity for Eu 3 þ
or Tb 3 þ increases significantly owing to the presence of the phosphate
which coordinates with the metal 140 ( Fig. 6.10C ). Several kinase biosensors
have been developed, including a kinase recognition domain for Src and
Abl, a carbostyril group that acts as a luminescence sensitizer, and an
iminodiacetate-chelating moiety that displayed up to 10-fold increase in sig-
nal upon phosphorylation 125 ( Fig. 6.10D ). The major limitations of this ap-
proach are that it requires the presence of free Tb 3 þ or Eu 3 þ as well as
photosensitizing groups coupled to the peptide biosensor and that it cannot
be applied in vivo because of the very short excitation wavelength (262 nm).
3.3. Biosensors involving quenching - unquenching strategies
3.3.1 Self-reporting biosensors
Aromatic amino acids, such as tyrosine and tryptophan, constitute good
quenchers of organic dyes through a process that involves p / p stacking be-
tween the aromatic groups. In particular, tryptophan has been used in a
countless number of assays to quench the fluorescence of probes. 141 A similar