The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through lightmediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.optogenetic tool | PER-ARNT-SIM domain | computational library | phage display | Rosetta molecular modeling suite

We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 ␤-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of ␤-lactam hydrolysis. Some of these MBP-BLA switches were effectively ''on-off'' in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth͞no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 ؋ 10 6 variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a ''sucrose-binding protein,'' illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.allostery ͉ ␤-lactamase ͉ maltose binding protein R egulation of protein activity is fundamental to cellular function.One of the mechanisms a cell uses to modulate the level of protein activity is regulation of the amount of a protein present in a cell. Accordingly, many strategies for engineering control of cellular protein activity have focused on modulation of protein production and degradation, often through small-moleculedependent switches that regulate transcription, translation, localization, degradation, or protein splicing (1) or through the engineering of artificial gene-regulatory networks (2). The key strength of many of these approaches is that they are easily generalized. For example, a switch that regulates transcription of one gene can easily be adapted to regulate transcription of an arbitrary gene. However, the significant limitation of these approaches is the slow dynamics stemming from the indirect nature of the regulation (i.e., protein activity is regulated by controlling the amount of protein and not by regulating the protein's specific activity directly). In addition, modulation is only feasible in the context of the cell and cannot be easily transferred to an in vitro setting.A more satisfying approach is to modulate the protein activity directly, but this presents a considerable design problem. Inhibitors, if they can be found, can only can be used to down-regulate activity, and such a strategy suffers from the fact that one is not free to choose the signal that modulates the activity: The signal must be an inhibitor of the protein. One clever way around this limitation is to engineer the inhibitor such that a third molecule can regulate the inhibitor's affinity for the regulated protein, as was demonstrated for RNA aptamer inhibitors that could be regulated by an organic small molecule (3). Natural allosteric proteins solve this problem by having spatially distinct regulatory and active sites. Ligand binding or covalent modifications at one site affects the output function at a distant site through a conformational change. This mechanism has ...

Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

We have created a molecular switch by the in vitro recombination of nonhomologous genes and subjecting the recombined genes to evolutionary pressure. The gene encoding TEM1 beta-lactamase was circularly permuted in a random fashion and subsequently randomly inserted into the gene encoding Escherichia coli maltose binding protein. From this library, a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose. Upon removal of maltose, RG13's catalytic activity returned to its premaltose level, illustrating that the switching is reversible. The modularity of RG13 was demonstrated by increasing maltose affinity while preserving switching activity. RG13 gave rise to a novel cellular phenotype, illustrating the potential of molecular switches to rewire the cellular circuitry.

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