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The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose-binding pocket of the maltose-activated β-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 β-lactamase into the E. coli maltose binding protein (MBP). We find that the maltose-dependent resistance to ampicillin conferred to the cells by the MBP317-347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22-fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30-fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch., (© 2016 The Protein Society.)

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.

Synthetic cellular logic gates are primarily built from gene circuits owing to their inherent modularity. Single proteins can also possess logic gate functions and offer the potential to be simpler, quicker, and less dependent on cellular resources than gene circuits. However, the design of protein logic gates that are modular and integrate with other cellular components is a considerable challenge. As a step toward addressing this challenge, we describe the design, construction, and characterization of AND, ORN, and YES logic gates built by introducing disulfide bonds into RG13, a fusion of maltose binding protein and TEM-1 β-lactamase for which maltose is an allosteric activator of enzyme activity. We rationally designed these disulfide bonds to manipulate RG13's allosteric regulation mechanism such that the gating had maltose and reducing agents as input signals, and the gates could be toggled between different gating functions using redox agents, although some gates performed suboptimally.

The ability to regulate cellular protein activity offers a broad range of biotechnological and biomedical applications. Such protein regulation can be achieved by modulating the specific protein activity or through processes that regulate the amount of protein in the cell. We have previously demonstrated that the nonhomologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in genes that confer maltose-dependent resistance to β-lactam antibiotics even though the encoded proteins are not allosteric enzymes. We showed that these phenotypic switches-named based on their conferral of a switching phenotype to cells-resulted from a specific interaction with maltose in the cell that increased the switches cellular accumulation. Since phenotypic switches represent an important class of engineered proteins for basic science and biotechnological applications in vivo, we sought to elucidate the phenomena behind the increased accumulation and switching properties. Here, we demonstrate the key role for the linker region between the two proteins. Experimental evidence supports the hypothesis that in the absence of their effector, some phenotypic switches possess an increased rate of unfolding, decreased conformational stability, and increased protease susceptibility. These factors alone or in combination serve to decrease cellular accumulation. The effector functions to increase cellular accumulation by alleviating one or more of these defects. This perspective on the mechanism for phenotypic switching will aid the development of design rules for switch construction for applications and inform the study of the regulatory mechanisms of natural cellular proteins., (Copyright © 2013 The Protein Society.)