Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions. Issue 17 (9th August 2019)

• Record Type: Journal Article
• Authors: Tichy, Alexandra-Madelaine; Gerrard, Elliot J.; Legrand, Julien M.D.; Hobbs, Robin M.; Janovjak, Harald
• Is Part Of: Journal of molecular biology. Volume 431:Issue 17(2019)
• Page Start: 3046
• Page End: 3055
• Publication Date: 2019-08-09
• Subjects: Optogenetics -- light-sensitive domain -- photoreceptor -- protein-protein interaction -- caspase9; LSD light-sensitive domain -- LOV light-oxygen-voltage sensing -- CRY cryptochrome -- PHY phytochrome -- CBD cobalamin binding domain -- PPI protein–protein interaction -- PHR photolyase homology region -- P2A porcine teschovirus-1 2A -- PCR polymerase chain reaction -- RT room temperature -- casp9 caspase-9; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2019.05.033
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo . We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology. Graphical abstract: Unlabelled Image Highlights: Optogenetic tools permit the spatio-temporal control of protein and cell function. A new method efficiently prepares modular optogenetic tools. The method combines a genetic engineering strategy and photoreceptor library. Using the strategy and library, light-activated caspases were developed. Optical regulation of diverse target proteins is now more directly accessible.