thus strongly influence change in the spectral properties of the cpFPs. The influence
is usually brought by factors such as protonation/deprotonation of a GFP-like
chromophore, as well as changes in its fluorescence quantum yield and molar
extinction coefficient. A rationally engineered cpGFP offers robust variants which
sustains fluorescence emission even after insertion of peptides or proteins to the new
terminus. The predominant permuted variants as per GFP sequence arise from a
permutation point in the vicinity of amino acid positions [62, 66]. The proximity of
sensitive fusion domains to the chromophore pocket in the fluorescent protein
determines extent of influence on the native spectral properties. In the case of
cpECFP, cpEGFP, and cpEYFP, insertion of calmodulin (Ca
2+ -binding protein) in
a specific position localized it in close proximity to the chromophore in the folded
3D conformation, which resulted in deprotonation of the chromophore and subsequent shift in fluorescence emission [60, 110]. As a follow-up to this pioneering
study, several groups developed single cpFP-based biosensors with different binding
domains for the detection of calcium [54, 111, 112], cGMP [113], H 2 O 2 [57], and
Zn(II) [114].
In cpFPs, the recognition domain in the presence of an analyte can undergo a
conformational change by itself as well as influence conformational change in the
fused fluorescent protein that is reflected by change in the fluorescence spectra. One
example of this approach is a G-CaMP sensor for Ca
2+ , which has a calmodulin
(Ca
2+ -binding protein) fused to the C-termini of a cpEGFP and a M13 peptide
(a synthetic peptide with calmodulin-binding domain) fused to the N termini
[115]. The success of the G-CaMP biosensor design inspired further modification
for improving the sensitivity while developing a diverse range of color palettes for
multicolor imaging of Ca
2+ level in different organelles of cells, such as cytosol,
nucleus, and mitochondria, at single-cell level [116].
The routine approach of permutation in FPs involves fusion of sensitive domains
close to the chromophore in order to manifest a change in its spectral properties
[77, 117]. With this strategy, numerous calcium sensors [55, 112, 118, 119] and
hydrogen peroxide sensors [57], phosphorylation sensors [120], and membrane
potential sensors [121, 122] have emerged successfully in the recent past. Incorporation of binding protein with competitive analyte affinity can serve as a new type of
ratiometric sensor. Incorporation of adenylate binding protein GlnK1 with differential affinity for ATP and ADP into cpYFP could generate sensors with different
spectral properties depending upon the analyte ADP or ATP [123].
Hydrogen peroxide is an important signaling molecule, and a sensor specific
for detection of hydrogen peroxidase, i.e., HyPer, was designed based on yellow
cpFP incorporated into the H 2 O 2 -sensitive OxyR regulatory domain [124]. The
sensor detects submicromolar concentration of H 2 O 2 by selective oxidation of
OxyR residues and leads to change in yellow cpFP chromophore environment
with corresponding ratiometric change in fluorescence excitation spectrum, i.e.,
fluorescence ratio upon excitation at 420 nm with respect to excitation at 500 nm.
The sensor was later successfully modified for the detection of wounds using zebra
fish as a model organism [125].
164
U. K. Sukumar et al.
is usually brought by factors such as protonation/deprotonation of a GFP-like
chromophore, as well as changes in its fluorescence quantum yield and molar
extinction coefficient. A rationally engineered cpGFP offers robust variants which
sustains fluorescence emission even after insertion of peptides or proteins to the new
terminus. The predominant permuted variants as per GFP sequence arise from a
permutation point in the vicinity of amino acid positions [62, 66]. The proximity of
sensitive fusion domains to the chromophore pocket in the fluorescent protein
determines extent of influence on the native spectral properties. In the case of
cpECFP, cpEGFP, and cpEYFP, insertion of calmodulin (Ca
2+ -binding protein) in
a specific position localized it in close proximity to the chromophore in the folded
3D conformation, which resulted in deprotonation of the chromophore and subsequent shift in fluorescence emission [60, 110]. As a follow-up to this pioneering
study, several groups developed single cpFP-based biosensors with different binding
domains for the detection of calcium [54, 111, 112], cGMP [113], H 2 O 2 [57], and
Zn(II) [114].
In cpFPs, the recognition domain in the presence of an analyte can undergo a
conformational change by itself as well as influence conformational change in the
fused fluorescent protein that is reflected by change in the fluorescence spectra. One
example of this approach is a G-CaMP sensor for Ca
2+ , which has a calmodulin
(Ca
2+ -binding protein) fused to the C-termini of a cpEGFP and a M13 peptide
(a synthetic peptide with calmodulin-binding domain) fused to the N termini
[115]. The success of the G-CaMP biosensor design inspired further modification
for improving the sensitivity while developing a diverse range of color palettes for
multicolor imaging of Ca
2+ level in different organelles of cells, such as cytosol,
nucleus, and mitochondria, at single-cell level [116].
The routine approach of permutation in FPs involves fusion of sensitive domains
close to the chromophore in order to manifest a change in its spectral properties
[77, 117]. With this strategy, numerous calcium sensors [55, 112, 118, 119] and
hydrogen peroxide sensors [57], phosphorylation sensors [120], and membrane
potential sensors [121, 122] have emerged successfully in the recent past. Incorporation of binding protein with competitive analyte affinity can serve as a new type of
ratiometric sensor. Incorporation of adenylate binding protein GlnK1 with differential affinity for ATP and ADP into cpYFP could generate sensors with different
spectral properties depending upon the analyte ADP or ATP [123].
Hydrogen peroxide is an important signaling molecule, and a sensor specific
for detection of hydrogen peroxidase, i.e., HyPer, was designed based on yellow
cpFP incorporated into the H 2 O 2 -sensitive OxyR regulatory domain [124]. The
sensor detects submicromolar concentration of H 2 O 2 by selective oxidation of
OxyR residues and leads to change in yellow cpFP chromophore environment
with corresponding ratiometric change in fluorescence excitation spectrum, i.e.,
fluorescence ratio upon excitation at 420 nm with respect to excitation at 500 nm.
The sensor was later successfully modified for the detection of wounds using zebra
fish as a model organism [125].
164
U. K. Sukumar et al.