Time resolved insights into the photo-activation mechanism of OCP by combined ultrafast optical spectroscopy and serial femtosecond crystallography. – DynOCP

The Orange Carotenoid Protein (OCP) is modular photoactive protein involved in cyanobacterial photoprotection. Strong blue light induces carotenoid and protein conformational changes causing a spectral shift and leading to carotenoid translocation and separation of two domains. These characteristics open the possibility to rationally engineer OCP variants suited for optogenetic applications. OCP would represent a useful addition to the optogenetic toolbox, adding not only a new class of chromophore but also a new type of triggering mechanism whereby initially coupled sensor and effector modules may reversibly or irreversibly dissociate. Moreover, it is possible to create reversible photoactive OCP-like from their isolated domains.
A prerequisite is the atomic level characterization of OCP photocycle and of its excited state dynamics that will lay the ground for the rational design of OCP-based photoswitches. In consequence, the aim of our project is to provide time-resolved structural insights into the photoactivation mechanism of OCP to create new OCP and OCP-like variants, rationally engineered to be used as photoswitches. To attain our objectives, we will use a combination of molecular biology, fast and ultra-fast transient absorption spectroscopy and time-resolved structural methods (TR- small-angle (SAXS) and wide-angle X-ray scattering (WAXS), and TR-serial crystallography (SX) at synchrotrons (SSX) and X-ray free electron lasers (SFX)). We devise a time-resolved visualization of photoactivation, from the formation of the excited-state structure following photon absorption on the few hundreds of fs timescale, to the migration of the carotenoid and (reversible/irreversible) dissociation of the two domains, on the ms timescale. First, we will perform a thorough TR spectroscopy characterization of WT and mutant OCPs, which will allow to identify the various states formed upon photo-illumination, and to determine their characteristic lifetimes. We will then seek to shed light on the excited structures of OCP, by performing TR-SFX experiments on the fs-ns time scale using in vitro and in vivo grown nanocrystals. Our results will inform about how the protein can be modified to increase it photoactivation quantum yield. The slower steps yielding to the photoactivated red state, occur on the µs-s time scale and are of large amplitude; this most probably will cause crystal explosion. Thus, other structural approaches (TR-SAXS/WAXS), not dependent on the crystalline state of the protein, will be used. The design of OCP variants will be first based on the knowledge acquired by Partner 2 laboratory over the past 10 years and then guided by data to be acquired in this project.
The success of DynOCP project requires the combination of complementary expertise and knowledge provided by the three French laboratories, LASIR, I2BC, IBS. Dr Michel Sliwa (LASIR) is an expert on TR fs spectroscopy of photo-active systems for photonic devices and biology. Dr Jacques-Phillipe Colletier (IBS) is active in the development of serial micro/nano-crystallography at synchrotrons and XFELs from sample-presentation approaches, to micro and nanocrystals formation and characterization, to software to sort diffraction data and interpret structural results. Dr Kirilovsky (I2BC) is a recognised specialist of light stress and light regulation, especially in the OCP related photoprotective mechanism that she discovered and characterized. Finally, the partners have already demonstrated success in securing beam-time and instrument-time at synchrotrons, X-ray free electron lasers and TR optical spectroscopy facilities over the last years. The successful of this project will have societal and socio-economic impacts since it will contribute not only to fundamental knowledge on the OCP photocycle, but also enable the rational development of OCP and OCP-like variants able to serve as optogenetic tools and as a light regulator in artificial photosynthetic systems.