GES, by virtue of being derived from naturally evolved proteins or protein components of cells, open the possibility for targeting them virtually to any compartment or
microcompartment of cells through fusion with an appropriate domain or by introducing short terminal peptides to form appropriate signal motifs [58–60]. The design of
fluorescent biosensors is based on a rationale of introducing a nimble manipulation of
target domains or fluorescent proteins, which involves conformational changes in the
spectral properties of fused domains or distance change, dipole orientation shift
between two proteins capable of FRET. FRET is a physical phenomenon in which a
donor fluorophore upon excitation transfers the energy non-radiatively to a neighboring
acceptor fluorophore, thereby causing the acceptor to emit its characteristic fluorescence of longer wavelength range [18]. Since FRET is highly sensitive to the distance
between donor and acceptor dipoles within the 1–10 nm range, they have become a
valuable tool to deduce biochemical events involving changes in molecular proximity,
such as protein-protein interactions, conformational changes in proteins, intracellular
ion concentrations, and enzyme activities [61].
The genetically encoded fluorescent sensors can be broadly categorized into four
groups according to the basic principles of the designs (Fig. 3):
1. Intrinsic environment sensitive fluorescent protein biosensor (single FP-based
sensors)
2. Engineered single FP-based sensors
(a) Incorporating a conformationally sensitive detector domain
(b) Circularly permuted FP sensors
3. FRET-based sensors containing two FPs
4. Translocation sensors/assays
Each of these sensor categories has distinct applications owing to their characteristics. Single native fluorescent proteins are the preferred option for ion sensors,
whereas engineered fluorescent proteins and two protein systems are better options
for deducing structural changes or a protein-protein FRET interaction, respectively.
3.1 Intrinsic Environment-Sensitive Fluorescent Protein
Biosensor (Single FP-Based Sensors)
As an intrinsic property of GFP and its derivatives, the spectral properties of the
chromophores are determined by the environment (i.e., pH or conformational
change). This property has been exploited to develop biosensors that measure pH
[53, 62, 63], halide anions [64], and redox potentials [65, 66]. The environmentinduced change within the chromophore pocket of fluorescent protein offers flexibility for generating variation in the fluorescence spectrum to probe analyte-specific
signal.
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microcompartment of cells through fusion with an appropriate domain or by introducing short terminal peptides to form appropriate signal motifs [58–60]. The design of
fluorescent biosensors is based on a rationale of introducing a nimble manipulation of
target domains or fluorescent proteins, which involves conformational changes in the
spectral properties of fused domains or distance change, dipole orientation shift
between two proteins capable of FRET. FRET is a physical phenomenon in which a
donor fluorophore upon excitation transfers the energy non-radiatively to a neighboring
acceptor fluorophore, thereby causing the acceptor to emit its characteristic fluorescence of longer wavelength range [18]. Since FRET is highly sensitive to the distance
between donor and acceptor dipoles within the 1–10 nm range, they have become a
valuable tool to deduce biochemical events involving changes in molecular proximity,
such as protein-protein interactions, conformational changes in proteins, intracellular
ion concentrations, and enzyme activities [61].
The genetically encoded fluorescent sensors can be broadly categorized into four
groups according to the basic principles of the designs (Fig. 3):
1. Intrinsic environment sensitive fluorescent protein biosensor (single FP-based
sensors)
2. Engineered single FP-based sensors
(a) Incorporating a conformationally sensitive detector domain
(b) Circularly permuted FP sensors
3. FRET-based sensors containing two FPs
4. Translocation sensors/assays
Each of these sensor categories has distinct applications owing to their characteristics. Single native fluorescent proteins are the preferred option for ion sensors,
whereas engineered fluorescent proteins and two protein systems are better options
for deducing structural changes or a protein-protein FRET interaction, respectively.
3.1 Intrinsic Environment-Sensitive Fluorescent Protein
Biosensor (Single FP-Based Sensors)
As an intrinsic property of GFP and its derivatives, the spectral properties of the
chromophores are determined by the environment (i.e., pH or conformational
change). This property has been exploited to develop biosensors that measure pH
[53, 62, 63], halide anions [64], and redox potentials [65, 66]. The environmentinduced change within the chromophore pocket of fluorescent protein offers flexibility for generating variation in the fluorescence spectrum to probe analyte-specific
signal.
156
U. K. Sukumar et al.