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    • Recently AFF was used to

    2023-01-28

    Recently, AFF was used to convert an FN3-based binder specific to the Src homology 2 (SH2) domain of cAbl kinase into an allosterically regulated, intermolecular fluorescent switch (Figure 2D). Binding to cAbl kinase co-operatively stabilized the assembly of the FN3-based binder from two partial fragments, and was resolved by intermolecular FRET between YFP and CFP [34]. This promises to be a generic method for converting globular binding scaffolds into allosteric fluorescent sensors. Genetically encoded, intramolecular FRET sensors incorporating FPs have yet to be developed, but their construction could be complicated by the fact that AFF-based allosteric receptors comprise partially unfolded proteins, which have loosely defined molecular states 32, 33. AFF was also used to engineer allosterically regulated actuators including a zymogen of barnase [35] and a protease-sensitive ratiometric GFP sensor [36]. The latter was constructed by duplicating the tenth C-terminal strand of GFP at the N terminus while separating it with a thrombin cleavage site; in addition, a point mutation was introduced in the native tenth C-terminal strand shifting the emission spectrum from green to yellow (Figure 2E). As a result, two duplicated strands that conferred yellow and green fluorescence competed for the same binding interactions with the GFP core; photo-assisted proteolytic cleavage of the native N-terminal strand subsequently enabled the C-terminal strand to associate permanently, resulting in a >2000-fold ratiometric dynamic fluorescence change. It is easily conceivable that the thrombin cleavage site could be replaced with allosteric binding receptors that stabilize one strand more than another in the presence of a target analyte. Such a design could lead to a new category of ratiometric fluorescent sensors with properties superior to those of intramolecular FRET sensors.
    Allosterically regulated enzymes for imaging, diagnostic, and therapeutic applications
    Allosteric protein switches as actuators of cellular functions
    Towards the bottom-up design of autonomously operating signaling systems A key goal of synthetic biology is to create tailor-engineered programmable Actinomycin D that autonomously sense, process, and respond to distinct molecular cues. This has been realized predominantly through top-down designs; for instance, by artificially rewiring key signaling nodes of modularly organized signaling pathways. In the simplest case, ectopic overexpression of G protein-coupled receptors (GPCR) was sufficient to reprogram input control of GPCR signaling and yield various designer cell-based therapeutics 76, 77, 78, 79. Alternatively, key regulatory and catalytic domains in the yeast mating pathway were interchanged to improve mating efficiency [80], and artificial protein–protein interactions (PPIs) were sufficient to reprogram input and/or output control of the MAPK pathway [81] and alter its signaling dynamics [82]. Synthetic PPIs were also used to reprogram two-component signaling pathways in E. coli, while artificial AI interactions reduced cross-activation of related two-component signaling pathways in the crowded environment of the bacterial plasma membrane [83]. Furthermore, the activity of the small GTPase Cdc42 was controlled through allosterically regulated GEFs, which were designed based on phosphorylation-sensitive peptide–PDZ autoinhibitory interactions (Figure 4E) [84]. Ultimately, bottom-up assembly strategies using well-characterized, genetically encoded protein-based switches that can be easily interfaced with defined cellular functions are desired. In particular, there is a need to develop enzyme-based signal transducing and actuating systems and expand the repertoire of synthetic protein-based parts beyond orthogonal PPI modules 85, 86. Highly specific viral proteases that have naturally evolved to operate in the complex environment of the cytosol may be well suited for this purpose. Protease-based switches have been developed that mediate biomolecular signals through the induced colocalization of constitutively active proteases 87, 88 or the assembly of split-protease mutants 89, 90, 91, 92, 93, 94, 95 in response to ligand binding, receptor dimerization, and post-translational modifications (Figure 4F). In addition, protease-based signals can be actuated through diverse mechanisms ranging from apoptosis, transcription, cleavage of a reporter protein, and protein degradation 96, 97. The latter can effectively reverse the proteolytic signal, which can limit noise by introducing negative feedback, and potentially enable the creation of more complex network motifs beyond linear signal amplification cascades [98].