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    • Second the choice of the PAABD is

    2023-01-31

    Second, the choice of the PAABD is critical for an efficient and sensitive biosensor. The sequence of the PAABD should present high affinity and efficient recognition of the phosphorylated substrate, as opposed to poor affinity for the unphosphorylated substrate, and should display a fully reversible behavior between both forms. Several modular phosphobinding domains (PBDs) have been well characterized, including Src-homology 2/3 (SH2 SH3) and phosphotyrosine-binding (PTB) domains, 14.3.3 proteins, forkhead-associated (FHA) domains, WW domains, WD40 domains, and the PBD of Plk1 (for reviews, see 83, 84, 85, 86, 87, 88, 89). The linker between these domains is generally designed to be flexible (i.e., rich in glycine, alanine, and proline residues), and its length has proved to be a critical factor in affecting FRET efficiency.90, 91, 92 Last but not least, the choice of the AFP/FRET pair is essential. Ideally, AFPs that do not photobleach too rapidly, that have high quantum yield, and that are red-shifted should be chosen to maximize FRET efficiency and minimize phototoxicity.93, 94 Komatsu et al. recently proposed an optimized backbone for genetically encoded FRET biosensors bearing the cyan fluorescent protein (CFP) or Turquoise as donors, the yellow fluorescent protein (YFP) or YPet as an acceptor fluorophore, and a very long linker connecting the ligand to the Triamcinolone australia domain, which basically abolishes dimerization of the FRET pair, and showed that this backbone was ideal for both kinase and GTPase FRET biosensors reporting on ERK, PKA, S6K, RSK, Ras, and Rac1.
    Fluorescent Peptide/Protein Biosensors Concerted efforts of chemists and biologists in designing fluorescent probes for biological applications have led to the development of a very different class of biosensors that do not rely on genetically encoded AFPs. Fluorescent peptide, polypeptide, or protein biosensors constitute attractive alternatives to genetically encoded biosensors in that they offer a high degree of versatility, yet also a high degree of control. This class of biosensors is engineered by exploiting peptide substrate sequences or protein domains that bind a specific analyte or interface, which serves as platforms for site-selective coupling of one or more fluorescent probe(s). The fluorescent probe may be coupled by different means to the peptide backbone—most often chemically, enzymatically, or through replacement of a fluorescent amino acid analog (for review, see Ref. 105). The design allows for freedom in the choice of the fluorescent probe, amongst a wide variety of wavelengths and synthetic probes, and for its incorporation at virtually any position within the peptide. Peptides and polypeptides offer several major advantages inherent to their nature. They can be readily produced through synthetic chemistry or recombinant protein engineering; are easy to handle, store, and characterize; and can further be modified with a wide variety of unnatural substituents, such as modified amino acids, to improve specificity and selectivity, as well as small synthetic fluorescent probes. Peptides are very small yet can serve as substrates, docking sequences, or complementary biomolecular recognition interfaces, thereby offering a wide array of possibilities and strong potential for development of fluorescent biosensors. Peptides also constitute scaffolds for a wide variety of technological improvements, including introduction of quenchers or caging of specific amino acids. Moreover, compared to antibodies, they are cheaper to produce, display low antigenicity, and are rapidly eliminated by the organism, thereby generating relatively low cytotoxicity. Peptide biosensors are readily applicable in vitro and in cell lysates. Their applicability in living cells and in vivo, however, remains challenging, requiring suitable methods to facilitate their intracellular delivery. Notwithstanding, major advances in the field of protein and peptide delivery over the past 20 years have provided efficient means of introducing this class of biomolecules into living cells and in vivo based on protein transduction domains and cell-penetrating peptides (CPPs).107, 108 Once the issue of delivery is solved, peptide biosensors offer a major advantage over genetically encoded biosensors, in that they allow for immediate and controlled use, compared to genetically encoded biosensors, which require long periods of time for their expression and/or maturation (Table 6.1).