Switchable proteins that can be regulated through exogenous or endogenous inputs have a broad range of biotechnological and biomedical applications. Switch creation is challenging, even more so for switches with multiple inputs. Here we describe the design of switchable enzymes that require both an effector molecule and an environmental condition 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. Structural and thermodynamic studies supported the hypothesis that switching resulted from an increase in conformational heterogeneity of the enzyme domain in the absence of effector. These results support the ensemble model of allostery and embody a general strategy for the rational design of protein switches including those with multiple inputs.

Rational design of protein switches based on the ensemble model of allostery

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.