title: Design of protein switches based on an ensemble model of allostery.
abstract: Switchable proteins that can be regulated through exogenous or endogenous inputs have a broad range of biotechnological and biomedical applications. Here we describe the design of switchable enzymes based on an ensemble allosteric model. First, we insert an enzyme domain into an effector-binding domain such that both domains remain functionally intact. Second, we induce 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 confirm the switching behaviour in vitro and in vivo. Structural and thermodynamic studies support the hypothesis that switching result from an increase in conformational entropy of the enzyme domain in the absence of effector. These results support the ensemble model of allostery and embody a strategy for the design of protein switches.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400N Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Jay H
authors.initials: JH
authors.lastname: Choi
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400N Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Abigail H
authors.initials: AH
authors.lastname: Laurent
authors.affiliation: Department of Biology, Johns Hopkins University, 3400N Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Hilser
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400N Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D019277:Entropy; D004798:Enzymes / Q000737:chemistry*; D066298:In Vitro Techniques; D058619:Maltose-Binding Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D004798:Enzymes; D058619:Maltose-Binding Proteins; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1