Engineering of Tunable Allosteric-like Fluorogenic Protein Sensors.

Optical protein sensors that enable detection of relevant biomolecules of interest play central roles in biological research. Coupling fluorescent reporters with protein sensing units has enabled the development of a wide range of biosensors that recognize analytes with high selectivity. In these sensors, analyte recognition induces a conformational change in the protein sensing unit that can modulate the optical signal of the fluorescent reporter. Here, we explore various designs for the creation of tunable allosteric-like fluorogenic protein sensors through incorporation of sensing protein units within the chemogenetic fluorescence-activating and absorption-shifting tag (FAST) that selectively binds and stabilizes the fluorescent state of 4-hydroxybenzylidene rhodanine (HBR) analogs. Conformational coupling allowed us to design analyte-responsive optical protein sensors through allosteric-like modulation of fluorogen binding.