Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 251

3.3.2 The deep quench strategy
The strategy described above is not applicable to residues that are not aro-
matic. Therefore, for Ser/Thr kinase biosensors, Lawrence and collaborators
developed the “deep quench” strategy, which involves shielding of the fluo-
rophore by a quencher in solution, which will be displaced upon phosphor-
ylation of the biosensor because of binding of a phosphorecognition domain
to the phosphorylated serine or threonine, which will in turn promote

A Self-reporting biosensor—tyrosine quenches probe fluorescence

P

OH

Tyr
phosphorylation

Peptide substrate

B Deep quench reporter

SH2 domain

Quencher

Ser/Thr
phosphorylation

P

OH

Peptide substrate

C Quenching through aggregation

Fatty acid

Peptide

P

P

Phosphorylation

P

P

P

Complex dissociation

P

P

P

Figure 6.11 Biosensors involving quenching - unquenching strategies. (A) Self-reporting
biosensor—the phosphorylatable tyrosine residue serves as a quencher that silences
the fluorescence of a proximal fluorophore; phosphorylation disrupts this interaction,
thereby leading to complete enhancement of fluorescence. 126-128 (B) Deep-quench
reporter developed for Ser/Thr peptides—fluorescence of a probe is quenched by a
compound in solution; phosphorylation promotes recruitment of a phosphoSer/Thr-
binding domain (e.g., 14-3-3) which promotes enhancement of probe fluorescence. 129,130
(C) Quenching through aggregation—an amphiphilic peptide biosensor composed of a
substrate domain with a phosphorylatable residue, labeled with fluorescein, and an
N-terminal carbon tail that promotes auto-assembly or aggregation of the peptides.
Peptide aggregation leads to fluorescence quenching within the micelle; phosphorylation
disrupts the micelle structure, thereby releasing the individual probes whose fluorescence
is enhanced. 131