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constituted by the AFP itself, whose spectral properties respond directly to
the target or analyte, as pH-dependent or redox-dependent AFP biosensors
( Fig. 6.2 ).
The most widely developed genetically encoded biosensors developed to
probe protein kinase activities are single-chain FRETbiosensors that report on
kinase activity through phosphorylation-induced changes in FRET between
A
C
Single-chain FRET biosensor
BIFC biosensor
Donor
Linker
Acceptor
Substrate
Substrate
Nterm AFP
Cterm AFP
Phosphorylation
FRET
P
P
Reconstituted
fluorescent AFP
Substrate
Substrate
D
Single AFP with exogenous sensing moiety
B
Two chain FRET biosensor
Donor
Acceptor
Substrate
Fluorescent AFP
Phosphorylation
E
Single AFP biosensor
FRET
P
Analyte
Substrate
Fluorescent AFP
Figure 6.2 Genetically encoded biosensors. Genetically encoded biosensors of kinase
activity are derived from at least one genetically encoded autofluorescent protein.
(A) Single-chain biosensors bear a pair of AFPs, within the same molecule, which are
brought together and undergo changes in fluorescence resonance energy transfer
due to intramolecular conformational changes in response to the phosphorylation
event. (B) Two-chain biosensors, in which two AFPs lie on two different molecules sus-
ceptible for interacting and undergoing intermolecular FRET when the two chains are
brought together for a protein/protein interaction. (C) Biosensors based on bimolecular
fluorescence complementation (BiFC), in which two fragments of a split AFP are brought
together as a consequence of target recognition, or in this case phosphorylation,
thereby reconstituting the intact and fully fluorescent protein. (D) Single-chain biosen-
sors composed of a single AFP whose spectral properties change in response to the rec-
ognition of a target by an exogenous sensing element (other than the AFP). (E) Single
AFP biosensors, whose spectral properties respond directly to the target or analyte, such
as pH-dependent or redox-dependent AFP biosensors.
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