Curated Optogenetic Publication Database

Abstract: The blue light photoreceptor cum transcription factors, aureochromes (Aureos), are present exclusively in photosynthetic stramenopiles. Co-existence of Light-Oxygen-Voltage (LOV) and basic leucine zipper (bZIP) is unique to Aureos - therefore ideal to study light-dependent DNA binding/transcriptional regulation. Further, Aureos' inverse effector-sensor topology, resembling several sensory eukaryotic transcription factors, makes them prototypical optogenetic scaffolds. In absence of 3D data, this study aims for a thorough investigation of the bZIP domains from Aureos and others, and their interaction with substrate DNA using tools from sequence/structural bioinformatics, network theory, molecular dynamics simulation and in vitro experiments. An in-depth comparison of 173 Aureo/plant/opisthokont bZIPs reveals Aureos' uniqueness and evolutionary significance in DNA binding specificity as well as dimer stability. An all-atom network analysis on representative bZIP-DNA co-crystal structures, especially the measurement of eigenvector centrality, further adds importance to hydrophobic interactions in the zipper region to stabilize bZIP dimer and facilitate DNA binding in Aureos and other bZIPs. The most notable finding is the unique presence of histidine at the basic region of Aureos unlike other bZIPs. Histidine not just promotes blue light independent substrate DNA-binding affinity but also serves as a potential switch point in Aureo/bZIP evolution.

Abstract: Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their counterparts from thermophilic microorganisms remain understudied. Here, we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber, MrLOV and MrLOVe, and show that they are photoactive, with recovery time values of 21 and 27 min, respectively, and thermostable. Crystal structures reveal that MrLOV, which lacks helices A'α and Jα, forms a parallel dimer, whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel dimers interacting via helices Jα. One MrLOVe dimer is symmetric, and the other is asymmetric, with conformational differences mirroring activation-related changes in other LOV domains. Our data provide the structural basis for understanding and engineering of thermophilic LOVs and pave the way for development of thermostable and photostable LOV-derived optogenetic tools and flavin-based fluorescent proteins.