No 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 remained 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.

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These ‘phenotypic switch’ genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.

The left-handed parallel beta-helix (LbetaH) is a structurally repetitive, highly regular, and symmetrical fold formed by coiling of elongated beta-sheets into helical "rungs." This canonical fold has recently received interest as a possible solution to the fibril structure of amyloid and as a building block of self-assembled nanotubular structures. In light of this interest, we aimed to understand the structural requirements of the LbetaH fold. We first sought to determine the sequence characteristics of the repeats by analyzing known structures to identify positional preferences of specific residues types. We then used molecular dynamics simulations to demonstrate the stabilizing effect of successive rungs and the hydrophobic core of the LbetaH. We show that a two-rung structure is the minimally stable LbetaH structure. In addition, we defined the structure-based sequence preference of the LbetaH and undertook a genome-wide sequence search to determine the prevalence of this unique protein fold. This profile-based LbetaH search algorithm predicted a large fraction of LbetaH proteins from microbial origins. However, the relative number of predicted LbetaH proteins per specie was approximately equal across the genomes from prokaryotes to eukaryotes.