Applications for protein switches are numerous In diagnostic
Applications for protein switches are numerous. In diagnostics, protein switches can detect analytes as components of inexpensive homogeneous assays that do not require specialized equipment or time-consuming incubation and washing steps characteristic of immunoassays . Protein switches have also proven invaluable in the quantitative imaging of molecular processes in Gedunin . In addition, protein switches can control the activity of key signaling proteins by non-invasive means, such as with light or biochemically inert ligands, which act orders of magnitude faster than inducible gene expression-based systems.
Here, we review recent progress in the construction of protein-based switches for monitoring and actuating molecular and cellular functions while identifying aspects critical for their successful design. Overall, this should greatly facilitate the challenging task of constructing protein-based switches, which has so far proven intractable to computational design methods 10, 11 (Box 1).
Allosteric fluorescent protein switches Genetically encoded Ca2+ sensors constitute the first generation of protein switches that exploited fluorescent proteins (FPs, see Glossary) for generating a measureable read-out 12, 13. Allosteric binding receptors for Ca2+ were generated by fusing calmodulin (CaM) to a CaM-binding peptide (CaM-BP) derived from the myosin light chain kinase. As CaM binds Ca2+, CaM-BP associates with CaM, causing the receptor to transition from an extended to a compact state. In the original design, this conformational change was detected through distance-dependent changes in the efficiency of fluorescence resonance energy transfer (FRET) between a yellow- (YFP) and cyan-fluorescent protein (CFP) . In a more integrated design, the Ca2+ receptor was inserted internally into GFP, resulting in Ca2+-triggered modulation of GFP fluorescence intensity 13, 14. Iterative improvements over the course of 15 years have yielded highly sensitive calcium sensors that can detect the comparatively small calcium bursts that occur during axon potential firing in neurons 15, 16. Based on these initial blueprints, more than 100 intramolecular FRET sensors have been developed to monitor a variety of molecular queues, ranging from protein–peptide, antibody–epitope, and protein–small molecule interactions to the detection of proteolysis, post-translational modifications, and mechanical properties, such as tension at cell–cell junctions 17, 18, 19. Biophysical studies demonstrated that intramolecular FRET sensors with the largest signal change exploit the natural propensity of FPs to dimerize, which enables them to toggle between two defined molecular states in the presence and absence of a molecular queue. This sets them apart from FPs that do not form such interactions and exist in poorly defined conformational and spectral ensembles . In practice, this requires the construction of linkers that are sufficiently rigid to counteract the dimerization of FPs in the absence of a molecular queue, but sufficiently loose to ensure efficient FRET and folding of the fluorophores.
Allosteric fluorescent protein switches based on mutually exclusive binding interactions Given that only a few naturally occurring protein families exhibit conformational changes sufficiently large for sensor construction, allosteric binding receptors frequently have to be engineered. Here, modular design strategies that utilize generic receptor modules and minimize the need to engineer protein-based binders de novo are preferred.
Allosteric fluorescent protein switches based on partially unfolded protein fragments Beyond the movement of structurally well-defined protein domains, many naturally occurring allosterically regulated protein functions rely on partially unfolded and intrinsically disordered protein fragments . Equivalent synthetic protein switches can be artificially engineered by means of alternative frame folding (AFF). Here, a portion of a protein that makes contact with a desired target ligand is duplicated and fused to the opposite end of the molecule, yielding a continuous polypeptide that equilibrates between native and circularly permuted conformations (Box 2)  while leaving parts of the protein unfolded. Proteins are turned into allosteric binding receptors by point mutations that selectively stabilize the circularly permuted conformation while destabilizing the native conformation upon ligand binding (Figure 2C). Both calbindin and a ribose-binding protein (RBP) were converted by AFF into Ca2+- and ribose-specific allosteric receptors, while ligand-induced conformational changes in the two receptors were detected through distance-dependent intramolecular FRET between two chemically conjugated fluorophores 32, 33.