(6ft) Rational design of multi-input controlled protein switches towards the development of target specific therapeutic enzymes and biosensors

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 and therapeutic 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.  A modular design of protein switches allows the possibility of developing versatile protein switches, in which different input domains can be coupled to the output domain.  Thus, protein switches that can be regulated through exogenous or endogenous inputs have a broad range of biotechnological and biomedical applications such as the next generation therapeutic enzymes and biosensor systems.  For enhanced target specific controls and improved specificity of the engineered protein switch, it is desirable to establish a means to regulate protein activity with multiple input controls; yet, engineering of such a complex regulatory system is challenging.  Here we describe the design of switchable enzymes that requires multi-input signals such as an effector molecule, temperature, pH, redox agent, or electrochemical signal for activation.  First, we inserted an enzyme domain into an effector-binding domain such that both domains remained functionally intact.  Second, we induced the fusion to behave as a switch through the introduction of a linker designed to respond to different environmental conditions including temperature, pH, or redox potential.  We confirmed the multi-input switching behavior in vitro and in vivo.  We also demonstrated that some of these engineered switches could function as protein logic gates.  Structural and thermodynamic studies supported the hypothesis that switching resulted from manipulation of allosteric control mechanism via change in conformational equilibria or dynamics of the fusion domains.  These results embody a general strategy for the rational design of complex protein switches with multi-level controls.