Prof. Gebhard Schertler (ETHZ and PSI) presents "SwissFEL adventures in room-temperature crystallography: From the structure of vertebrate and invertebrate rhodopsins to new applications in optogenetics".
Abstract: We explore the use of light switchable G protein-coupled receptors (GPCRs) for the development of new optogenetic tools to control cellular signalling processes using light: OptoGPCRs. In a first stage, we identified several new opsins triggering GPCR signalling pathways. We extensively characterized the most promising candidates of invertebrate opsins biochemically in cellular assays and finally in vivo. We developed the basis for engineering bistable opsins, light switchable opsins, towards more effective optogenetic tools that are applicable in a very wide range of physiological processes. We determined the structure of a recombinant invertebrate rhodopsin and we carried out a detailed study of the chromophore binding site with advanced biophysical and crystallographic methods. In the case of vertebrate rhodopsins, we were able to use SwissFEL to observe directly the retinal isomerization with room-temperature serial crystallography. We were able to compare in detail monostable and bistable visual pigments. The bistable pigments in several aspects are closer to the ligand binding pharmacologically relevant GPCRs. In a successful engineering attempt, we were able to identify mutations that shift the wavelengths of an invertebrate rhodopsin towards the infrared or UV. This is important for the depth of penetration of light into tissues and for the ability to switch singalling ON and OFF with different light colours. The engineered OptoGPCRs are an important alternative to the channel opsins related optogenetic tools. OptoGPCRs have a wide range of applications that are not restricted to neurons. In future work, we can include the information we gained from single particle cryo-EM studies of rhodopsin / G protein singalling complexes. This will enable us to engineer the signalling output of the newly designed OptoGPCRs. These new tools have the potential to revolutionize many biologically important areas of medical science.