The progress made by our consortium has significantly advanced the field of optogenetics and GPCR signalling beyond the current state of the art. Our research has uncovered fundamental insights into the retinal isomerization of a novel protein -bestrhodopsin - using the most advanced spectroscopic methods available. These technologies are being directly applied to our novel optoGPCR targets, including the chimera constructs, which we have successfully developed.
We have also adapted time-resolved cellular assays initially developed for melanopsin to our bistable pigment targets such as invertebrate rhodopsins. These assays proved essential for characterizing an invertebrate rhodopsin mutant with good spectral separation, which will facilitate the characterization of all engineered bistable rhodopsins. This effort is crucial for the progress of the SOL project, and we are currently on track to meet our objectives.
A major milestone in our research is determining the structure of an active invertebrate rhodopsin. This breakthrough has deepened our understanding of the relationship between G proteins in invertebrates and vertebrates, leading to the development of a new tool for structural biology that enhances the efficiency of cryo-EM studies for bistable rhodopsins. Furthermore, we have explored the use of retinal analogues as super agonists, successfully integrating this strategy into our toolbox. Our structural work has led to immediate impacts on our protein engineering approach, resulting in the creation of a fusion protein to activate desired signalling pathways. We have combined colour-shifting mutations with GPCR fusions, yielding promising proof of bistability for this construct in vitro and in cellular assays.
Additionally, we have extended our strategy to include various constructs targeting cone pigments, alongside novel targets like insect rhodopsin and parapinopsins. Our research on crustacean rhodopsins has evolved from shrimp to well-expressing fish louse pigments, integrating the latest findings. In collaboration with Japanese teams, we have supported studies on coral opsins, helping to solidify proposal that chloride acts as a counterion.
We develop an AI approach for wavelength prediction for retinal proteins, which shows excellent potential for both microbial rhodopsins and bistable pigments. We have also employed QM/MM methods to calculate absorption maxima for visual pigments. In a remarkable simulation of polyene isomerization conical intersections, they provided detailed ab initio calculations that illustrated how the surrounding environment influences the reaction mechanism based on the shape of the conical intersection. Overall, this progress is expected to yield significant advancements in understanding and manipulating bistable rhodopsins and GPCR signalling, pushing the boundaries of optogenetics applications.
