In this section, we will describe these strategies GW 572016 in more detail, highlighting the insights from the practical standpoint.2.1. Dual AFP-Fused FRET-Based BiosensorsFRET is a physicochemical phenomenon which only occurs when two fluorophores are in sufficient proximity (<10 nm) of each other, and the emission spectrum of the donor overlaps the excitation spectrum of the acceptor [57]. In the AFP-based FRET strategy, CFP and YFP mutants have been favorably utilized as a FRET donor and an acceptor, respectively [58]. Engineering with two AFPs in combination with the receptor protein affords a sensor protein that responds to dynamic fluctuation of intracellular ligand concentration by a ratiometric fluorescence change. The feasibility of this strategy strongly depends on the magnitude of the structural change of the receptor.

In the case of the receptor with a large structural change upon binding the substrate, Inhibitors,Modulators,Libraries this strategy would be the most straightforward way to integrate the signal transduction function into the receptor of interest (Figure 1a), Inhibitors,Modulators,Libraries although serious concerns have been pointed out that the obtained FRET signals do not simply reflect the change in the expected distance of FRET pairs [58,59]. The trailblazing work for this sensor was reported by Miyawaki et al., in which a genetically encoded calcium indicator composed of two different colored AFP mutants and calmodulin, a Ca2+ receptor, Inhibitors,Modulators,Libraries has been constructed [36].Figure 1.Schematic illustration shows a concept of ligand sensing by dual AFP-fused FRET-based biosensors.

Currently, CFP and YFP mutants are preferentially selected as FRET donor and acceptor, respectively. (a) Intramolecular FRET-based biosensors exploit the …The receptor complex that accompanies the dissociation or the association of multiple subunits upon ligand binding was also suitable for Inhibitors,Modulators,Libraries the design of FRET-based biosensors (Figure 1b). Zaccoro and coworkers constructed a ratiometric fluorescent biosensor for cyclic adenosine monophosphate (cAMP) on the basis of an intermolecular FRET system between regulatory (R) and catalytic (C) subunit of protein kinase A (PKA) [47,48]. This biosensor can detect the rise of intracellular cAMP concentration by the decrease in the FRET efficiency induced by the dissociation of C subunit from R subunit. More Dacomitinib comprehensive information on dual AFP-fused FRET-based biosensors is available in other excellent reviews [60�C62].

Recently, Johnsson and coworkers have devised another class of FRET-based semisynthetic biosensors for Zn2+ combined with an AFP and a synthetic fluorophore based on the SNAP-tag labeling Vandetanib cancer technology [63]. Because SNAP-tag fusion protein can be covalently modified with O6-benzylguanine derivatives in living cells [64], the ap
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