Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.

Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2-CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2-CIB system by identifying versions of CRY2-CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13-α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains.

Necroptosis is a form of programmed necrotic cell death in which a signaling cascade induces oligomerization of mixed lineage kinase domain-like (MLKL) protein, leading to plasma membrane rupture. Necroptotic cell death is recognized as important for protection against viral infection and has roles in a variety of diseases, including cancer and diabetes. Despite its relevance to health and disease states, many questions remain about the precise mechanism of necroptotic cell death, cellular factors that can protect cells from necroptosis, and the role of necroptosis in disease models. In this study, we engineered a light-activated version of MLKL that rapidly oligomerizes and is recruited to the plasma membrane in cells exposed to light, inducing rapid cell death. We demonstrate this tool can be controlled spatially and temporally, used in a chemical genetic screen to identify chemicals and pathways that protect cells from MLKL-induced cell death, and used to study signaling responses of non-dying bystander cells. In additional studies, we re-engineered MLKL to block its cell-killing capacity but retain light-mediated membrane recruitment, developing a new single-component optogenetic tool that allows modulation of protein function at the plasma membrane.

Reprogramming the Cleavage Specificity of Botulinum Neurotoxin Serotype B1.

Proteases with reprogrammed specificity for nonnative substrates are highly desired in synthetic biology and biomedicine. However, generating reprogrammed proteases that are orthogonal and highly specific for a new target has been a major challenge. In this work, we sought to expand the versatility of protease systems by engineering an orthogonal botulinum neurotoxin serotype B (BoNT/B) protease that recognizes an orthogonal substrate. We designed and validated an orthogonal BoNT/B protease system in mammalian cells, combining mutations in the protease with compensatory mutations in the protease substrate and incorporating a truncated target sequence and then demonstrated use of this orthogonal BoNT/B protease-substrate combination to regulate complex transcriptional circuitry in mammalian cells. Transposing this platform into yeast, we demonstrated utility of this approach for in vivo protease evolution. We tested this platform with the newly designed orthogonal protease and then used it in a high-throughput screen to identify novel orthogonal protease/protease substrate combinations. While carrying out this work, we also generated new cleavage reporters that could be used to report botulinum toxin protease activity in mammalian cells using simple fluorescent readouts. We envision that these approaches will expand the applications of botulinum protease in new directions and aid in the development of new reprogrammed proteases.

An optimized optogenetic clustering tool for probing protein interaction and function.

The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, 'CRY2olig', which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, Light-Induced Co-clustering, that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.

Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p.

We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane (TM) segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Cross-linked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of cross-linking between TM segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the TM segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that TM segment 6, but not TM segment 5, exhibits rotational mobility that is not associated with receptor activation.