title: Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Amir
authors.initials: A
authors.lastname: Taslimi
authors.affiliation: Department of Chemistry, Southern Methodist University, Dallas, Texas, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Zoltowski
authors.affiliation: Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Jose G
authors.initials: JG
authors.lastname: Miranda
authors.affiliation: Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Gopal P
authors.initials: GP
authors.lastname: Pathak
authors.affiliation: Department of Biology, Duke University, Durham, North Carolina, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Hughes
authors.affiliation: Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Chandra L
authors.initials: CL
authors.lastname: Tucker
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056931:Cryptochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D062308:Optogenetics / Q000379:methods*; D011485:Protein Binding / Q000528:radiation effects; D000072417:Protein Domains / Q000528:radiation effects; D015202:Protein Engineering; D055503:Protein Multimerization / Q000528:radiation effects*; D015398:Signal Transduction / Q000528:radiation effects
chemical_list: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D056931:Cryptochromes; D019426:Integrases; C533832:CIB1 protein, Arabidopsis; C103618:CRY2 protein, Arabidopsis; C045073:Cre recombinase