another group introduced a metal-binding amino acid, HqAla (2-amino-3(8-hydroxyquinolin-5-yl)propanoic acid), into the Tyr66 of a cpsfGFP variant that
enabled this construct to achieve a 7.2-fold increase in fluorescence intensity in
the presence of Zn
2+ ions in living E. coli cells [102]. Apart from affinity-based
unnatural amino acids, chemically reactive UAAs also serve as an option for
reaction-based fluorescent protein biosensors. Schultz and coworkers exploited this
approach in designing an FP sensor (UFP-Tyr66pBoPhe) for detection of H 2 O 2 by
substituting Tyr66 of GFP with p-borono-L-phenylalanine (pBoPhe) carrying an
H 2 O 2 -reactive arylboronate side chain [102]. In the absence of H 2 O 2 , the chromophore remains electron deficient owing to the presence of electron withdrawing
vacant 2p orbital of boron, and as a result of which, the sensor does not produce
fluorescence. However, in the presence of H 2 O 2 , pBoPhe is oxidized to the original
tyrosine residue, leading to quick recovery of fluorescence [103]. Although initially
UAA-based sensors were speculated to behave unsuccessfully in in vivo systems
owing to the synthetic origin of UAA, a few recent reports have achieved the same
in mammalian cells. An UAA-based H 2 S sensor was developed by substitution of
Tyr66 with p-azido-L-phenylalanine (pAzF) and was successfully expressed in
mammalian cells and illustrated as response time of mere ~7 min upon addition of
50 μM of NaHS [104]. The azide-modified chromophore in the presence of H 2 S is
selectively reduced which results in the observed fluorescence enhancement. The
same group also developed genetically encoded mammalian cells compatible for
peroxynitrite probe on the basis of the similar strategy [105].
The oligomeric aspect of fluorescent proteins offers flexibility for designing
dimerization-dependent fluorescent sensors that also enables reversible fluorescence
change upon complementation [106]. For instance, the oligomeric Discosoma red FP
(DsRed) and the RFP heterodimer (ddRFP-A1B1) exhibit weak fluorescence in the
monomeric state but upon heterodimerization manifest tenfold higher fluorescence
with a Kd of 33 μM. A series of red intensiometric biosensors based on a diverse
color palette ddRFP, ddGFP, and ddYFP have been created for detection of PPIs,
Ca
2+ dynamics, and protease activity with improved brightness and contrast [107].
The efficient SNR of the system enabled imaging of endomembrane proximity
between endoplasmic reticulum and mitochondria clearly distinguishable.
3.2.2 Biosensors Designed Using Circularly Permuted FPs
The close proximity of N- and C-termini observed in many three-dimensional
protein structures has been used in the past to perform circular permutation experiments on many different proteins [108]. The circularly permuted FP (cpFP)-based
GES are quite promising owing to the potentially high dynamic range of fluorescence spectral shifts. A circular permutation is a relationship between proteins
whereby they have a changed order of amino acids in their peptide sequence
resulting in a reconstituted protein with overall similar 3D shape but with different
N- and C-termini [109]. In the case of cpFP-based sensors, conformational changes
of sensory domains yield structural changes in the chromophore environment and
Applications of Fluorescent Protein-Based Sensors in Bioimaging
163
enabled this construct to achieve a 7.2-fold increase in fluorescence intensity in
the presence of Zn
2+ ions in living E. coli cells [102]. Apart from affinity-based
unnatural amino acids, chemically reactive UAAs also serve as an option for
reaction-based fluorescent protein biosensors. Schultz and coworkers exploited this
approach in designing an FP sensor (UFP-Tyr66pBoPhe) for detection of H 2 O 2 by
substituting Tyr66 of GFP with p-borono-L-phenylalanine (pBoPhe) carrying an
H 2 O 2 -reactive arylboronate side chain [102]. In the absence of H 2 O 2 , the chromophore remains electron deficient owing to the presence of electron withdrawing
vacant 2p orbital of boron, and as a result of which, the sensor does not produce
fluorescence. However, in the presence of H 2 O 2 , pBoPhe is oxidized to the original
tyrosine residue, leading to quick recovery of fluorescence [103]. Although initially
UAA-based sensors were speculated to behave unsuccessfully in in vivo systems
owing to the synthetic origin of UAA, a few recent reports have achieved the same
in mammalian cells. An UAA-based H 2 S sensor was developed by substitution of
Tyr66 with p-azido-L-phenylalanine (pAzF) and was successfully expressed in
mammalian cells and illustrated as response time of mere ~7 min upon addition of
50 μM of NaHS [104]. The azide-modified chromophore in the presence of H 2 S is
selectively reduced which results in the observed fluorescence enhancement. The
same group also developed genetically encoded mammalian cells compatible for
peroxynitrite probe on the basis of the similar strategy [105].
The oligomeric aspect of fluorescent proteins offers flexibility for designing
dimerization-dependent fluorescent sensors that also enables reversible fluorescence
change upon complementation [106]. For instance, the oligomeric Discosoma red FP
(DsRed) and the RFP heterodimer (ddRFP-A1B1) exhibit weak fluorescence in the
monomeric state but upon heterodimerization manifest tenfold higher fluorescence
with a Kd of 33 μM. A series of red intensiometric biosensors based on a diverse
color palette ddRFP, ddGFP, and ddYFP have been created for detection of PPIs,
Ca
2+ dynamics, and protease activity with improved brightness and contrast [107].
The efficient SNR of the system enabled imaging of endomembrane proximity
between endoplasmic reticulum and mitochondria clearly distinguishable.
3.2.2 Biosensors Designed Using Circularly Permuted FPs
The close proximity of N- and C-termini observed in many three-dimensional
protein structures has been used in the past to perform circular permutation experiments on many different proteins [108]. The circularly permuted FP (cpFP)-based
GES are quite promising owing to the potentially high dynamic range of fluorescence spectral shifts. A circular permutation is a relationship between proteins
whereby they have a changed order of amino acids in their peptide sequence
resulting in a reconstituted protein with overall similar 3D shape but with different
N- and C-termini [109]. In the case of cpFP-based sensors, conformational changes
of sensory domains yield structural changes in the chromophore environment and
Applications of Fluorescent Protein-Based Sensors in Bioimaging
163