EnvELOP: Environmental tuning of microbial rhodopsins Electronic Landscape for New Optogenetic Potentialities

The emerging field of optogenetics exploits the genetic encoding of photoactive proteins to control physiological processes: the final goal is to switch on and off specific molecular events in a controlled way, with a high spatiotemporal resolution and in a wavelength-dependent fashion. Microbial rhodopsins (mRh) are the main tools of optogenetics as their light-gated channel or light-driven pumping functions are used to regulate neuronal activity with light. Protein engineering of mRh, and in particular structure-guided mutagenesis, has only been partially successful in the design of optogenetic devices with improved properties: their potential applications are thus far from being fully realized and key limitations still exist. The present inability to overcome such limitations mainly arises from the lack of understanding of the relationship between structure and molecular mechanism in mRhs, as a prerequisite for the engineering of the next generation of optogenetics tools. In this proposal we focus on the role of the chromophore binding cavity hydrogen bonding network (HBN) as a critical but substantially unexplored structural To this end, we develop and apply a theoretical-computational factor affecting the mRh photochemistry. machinery able to rationalize the light activation step in different microbial rhodopsins as a function of their HBN variations. More in general, EnvELOP will focus on the systematic investigation of a suitable set of wild-type mRhs and of their mutants to establish relationships between structural changes (in terms of HBN, chromophore cavity dimension, protein environment effects) and mRh photoreactivity (in terms of absorption wavelength, excited state lifetime, photoisomerization dynamics). These quantities have been shown to be substantially different from one mRh to another, yet the origin of these differences is still unclear. EnvELOP proposes an effective method to simulate infrared spectra before and after the photoisomerization and to access atomistic and mechanistic information on the effects of different HBNs on mRh photochemical reactivity. To gain insights into the HBN regulated photoisomerization process and the associated excited state lifetime, we will implement an original multireference QM/MM methodology that includes mutual polarization and dispersion interactions between the protein and the chromophore in an automated mRhs model building generator that would make it possible to investigate full sets of variants in a systematic and highly informative fashion.