few to highlight here include a hyperthermophilic protein [24], two small molecule
biosensor proteins [25], two novel enzymes [26, 27], and a novel protein fold
[28]. Thus, computational in-silico approach holds great promise in future for
designing and optimization of biosensors. While developing mutants for designing
biosensors, it is also important to consider the use of infrared and near-infrared (NIR)
fluorescent proteins as a choice for enhancing the in vivo imaging capability of the
developed biosensors.
Green fluorescent protein (native state) is a 21 kDa protein consisting of
238 amino acid residues forming a secondary structure of 5 α-helices and 1
11-stranded β-pleated sheet, where each strand contains 9–13 amino acid residues
each [29]. Substitution of specific amino acids has generated a wide range of GFP
variants with distinct spectral characteristics. For instance, substituting Tyr66 for
His, Trp, or Phe results in blue-shifted spectral variants. Extensive mutagenesis of
Aequorea victoria GFP has produced a series of monomeric FPs of a variety of
colors: blue [30, 31], violet [32], cyan [30, 31, 33, 34], green [35–37], and yellow
[38]. This palette enables multicolor labeling of proteins of interests and FRETbased techniques. Breakthrough in the red fluorescent protein field occurred only
after the discovery of DsRed and other red fluorescent and chromoproteins from
Anthozoa species [39–41]. These discoveries opened the way for the development of
orange, red, and far-red FPs with emission peaks located as far as 655 nm [42]. However, the vast majority of natural FPs and chromoproteins cloned from various
species during the past 10 years are tetramers, such as FPs from anthozoa [39–41]
and copepods [43, 44], or dimers, such as anm2CP and phiYFP from hydrozoa [44].
3 Genetically Encoded Sensors (GES)
The genetically encoded fluorescent proteins have opened new avenues for
developing biosensors to visualize and quantify activity or conformational state of
proteins of interest, especially changes in the concentration of molecular and physiological events in cells, tissues, or whole organism [45]. The intercellular/intracellular signaling pathways, cell communications, differentiation, and development
have been investigated extensively with these fluorescent proteins [46]. One of the
promising applications include in vivo imaging of individual neurons in transgenic
animals with calcium-responsive genetically encoded biosensors [47]. At the intracellular levels, genetically encoded biosensors can be used to spatiotemporally
decipher the complex network of interactions that occur between proteins, nucleic
acids, and other macromolecules (Fig. 2) [48]. The chemically synthesized sensitive
fluorescent dyes differ greatly from fluorescent proteins in terms of their relevance,
sensitivity, specificity, development, and applications. The genetically encoded
fluorescent protein sensors are introduced into the host cells as genetic materials
by either transient transfection or knock-in techniques which allows cellular endogenous biogenesis pathways to express these as proteins [49]. Such an intricate
integration with endogenous biogenesis eliminates the possibility of unintended
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U. K. Sukumar et al.
biosensor proteins [25], two novel enzymes [26, 27], and a novel protein fold
[28]. Thus, computational in-silico approach holds great promise in future for
designing and optimization of biosensors. While developing mutants for designing
biosensors, it is also important to consider the use of infrared and near-infrared (NIR)
fluorescent proteins as a choice for enhancing the in vivo imaging capability of the
developed biosensors.
Green fluorescent protein (native state) is a 21 kDa protein consisting of
238 amino acid residues forming a secondary structure of 5 α-helices and 1
11-stranded β-pleated sheet, where each strand contains 9–13 amino acid residues
each [29]. Substitution of specific amino acids has generated a wide range of GFP
variants with distinct spectral characteristics. For instance, substituting Tyr66 for
His, Trp, or Phe results in blue-shifted spectral variants. Extensive mutagenesis of
Aequorea victoria GFP has produced a series of monomeric FPs of a variety of
colors: blue [30, 31], violet [32], cyan [30, 31, 33, 34], green [35–37], and yellow
[38]. This palette enables multicolor labeling of proteins of interests and FRETbased techniques. Breakthrough in the red fluorescent protein field occurred only
after the discovery of DsRed and other red fluorescent and chromoproteins from
Anthozoa species [39–41]. These discoveries opened the way for the development of
orange, red, and far-red FPs with emission peaks located as far as 655 nm [42]. However, the vast majority of natural FPs and chromoproteins cloned from various
species during the past 10 years are tetramers, such as FPs from anthozoa [39–41]
and copepods [43, 44], or dimers, such as anm2CP and phiYFP from hydrozoa [44].
3 Genetically Encoded Sensors (GES)
The genetically encoded fluorescent proteins have opened new avenues for
developing biosensors to visualize and quantify activity or conformational state of
proteins of interest, especially changes in the concentration of molecular and physiological events in cells, tissues, or whole organism [45]. The intercellular/intracellular signaling pathways, cell communications, differentiation, and development
have been investigated extensively with these fluorescent proteins [46]. One of the
promising applications include in vivo imaging of individual neurons in transgenic
animals with calcium-responsive genetically encoded biosensors [47]. At the intracellular levels, genetically encoded biosensors can be used to spatiotemporally
decipher the complex network of interactions that occur between proteins, nucleic
acids, and other macromolecules (Fig. 2) [48]. The chemically synthesized sensitive
fluorescent dyes differ greatly from fluorescent proteins in terms of their relevance,
sensitivity, specificity, development, and applications. The genetically encoded
fluorescent protein sensors are introduced into the host cells as genetic materials
by either transient transfection or knock-in techniques which allows cellular endogenous biogenesis pathways to express these as proteins [49]. Such an intricate
integration with endogenous biogenesis eliminates the possibility of unintended
154
U. K. Sukumar et al.