Engineering strategy and vector library for the rapid generation of modular light-controlled protein–protein interactions Tichy, Alexandra-Madelaine Gerrard, Elliot J. Legrand, Julien M.D. Hobbs, Robin M. Janovjak, Harald L ; https://orcid.org/0000-0002-8023-9315 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. Elsevier 2019 info:eu-repo/semantics/article doc-type:article text http://purl.org/coar/resource_type/c_2df8fbb1 https://research-explorer.ista.ac.at/record/6564 Tichy A-M, Gerrard EJ, Legrand JMD, Hobbs RM, Janovjak HL. Engineering strategy and vector library for the rapid generation of modular light-controlled protein–protein interactions. <i>Journal of Molecular Biology</i>. 2019;431(17):3046-3055. doi:<a href="https://doi.org/10.1016/j.jmb.2019.05.033">10.1016/j.jmb.2019.05.033</a> eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2019.05.033 info:eu-repo/semantics/altIdentifier/issn/0022-2836 info:eu-repo/semantics/altIdentifier/e-issn/1089-8638 info:eu-repo/semantics/altIdentifier/wos/000482872100002 info:eu-repo/semantics/openAccess