Abstract
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.
Highlights
A biosensor consists essentially of an input module, responsible for interacting with the target molecule, and an output module which transforms the molecule recognition into a detectable signal [1]
Based on the previously identified nonallosteric bifunctional chimera where cpBLA was inserted into maltose-binding protein (MBP), the linker region was engineered to increase the conformational flexibility of the chimera in the absence of maltose
R-iGluSnFR0.1 and Rncp-iGluSnFR1, worked successfully as biosensors for glutamate when targeted to the surface of HEK-293 cells [67]. These results show that the changing of protein topology may offer a new diversity layer for engineering fluorescent biosensors
Summary
A biosensor consists essentially of an input module, responsible for interacting with the target molecule, and an output module which transforms the molecule recognition into a detectable signal [1]. A typical protein switch is a biomolecule that can change between two or more distinct conformations (or conformational ensembles) in response to a specific stimulus [11, 12] These changes modulate their active state – output − (e.g., enzyme activity, ligand affinity, fluorescence, and oligomeric state) in response to a binding event or physical signal – input – (e.g., small molecule, pH, covalent modification, and light). Protein switches are key components able to couple cellular functions Their behavior is similar to the natural allosteric proteins, exhibiting remarkable attributes that make them an extraordinary model to design biosensors, such as high specificity and affinity, reversible signal transduction, versatility, and fast response, acting in millisecond to microsecond timescale [13, 14] which is faster than inducible gene expression-based systems (seconds to hours) [15].
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