The Utilization of Conformational Entropy in the Design of Multi-Input Switches

Switchable proteins that can be regulated through exogenous or endogenous inputs have a broad range of biotechnological and biomedical applications.  The engineering of such switches is challenging, even more so for switches that have multiple input controls. Here we describe the design of switchable enzymes that require both an effector molecule and an environmental condition (temperature or pH) 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 conditional conformational flexibility designed to increase the conformational entropy of the enzyme domain in a temperature- or pH-dependent fashion.  We confirmed the multi-input switching behavior in vitro and in vivo. Protein unfolding and protein NMR studies supported the hypothesis that switching resulted from an increase in conformational dynamics of the enzyme domain in the absence of effector. These results embody a general strategy for the rational design of complex protein switches with multi-level control.