Engineered Multi-Input Protein Switches As Versatile Biosensors with Target-Specific Controls

Engineered multi-input protein switches as versatile biosensors with target-specific controls

 

Jay H. Choi and Marc Ostermeier

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland

Protein switches are engineered proteins that are composed of an input domain that recognizes and responds to an input signal and an output domain whose function is regulated by the state of the input domain.  Engineered protein switches have a number of exemplary properties for sensing applications including a large dynamic range, high specificity for the activating ligand, and a modular architecture that will facilitate fine-tuning of the desired properties.  Thus, protein switches can provide a unique platform for the next generation biosensor systems.  The modular regulation mechanism of protein switches allows the possibility of developing versatile protein switches, in which different input domains can be coupled to the same output domain.  However, switches engineered to date are limited to have natural ligands or similar biomolecules as input signals.  For the development of versatile biosensors, it would be desirable to have input signals that can be easily controlled or to have multiple input signals that can be manipulated independently.  Especially in cellular environment, multi-regulated switches is highly desirable for the target specific control.  We have developed protein switches that can be simultaneously regulated by multiple input signals including small molecules, temperature, pH, and redox potential.  These protein switches are designed and constructed based on a platform of a previously developed small molecule activated protein switch.  We have taken advantage of its modular regulation mechanism that controls the activation of enzyme in the output domain by manipulating active conformational states.  These protein switches function via the dual regulation mechanisms such that they are pre-activated by changes in temperature, redox potential, or pH, and then subsequently activated by small molecules.  Thus, the engineered multi-regulated protein switches require dual input signals for the complete activation of enzyme.  This design principle has also been applied to develop reversely regulated protein switches.