Structural studies of the photosynthetic reaction center of Blastochloris viridis using XFEL and Synchrotron sources

Abstract

Life on Earth as we know it has been possible because of photosynthesis, a process by which hydrocarbons and chemical energy are generated using the energy extracted from sunlight. As with any other biological process, there is a protein responsible and it is called the reaction center. It is found in various photosynthetic organisms, including bacteria, algae, and plants. A photosynthetic reaction center has a conserved structure and function, where light absorption triggers an electron transfer process leading to energy production. This thesis is focused on the bacterial reaction center from Blastochloris viridis, analogous to photosystem II in plants. This study is focused on characterization of protein structural changes upon light absorption. For decades, X-ray crystallography has been a key method for determining protein atomic structures. X-ray free-electron lasers have facilitated new approaches, called serial femtosecond X-ray crystallography (SFX), which enable new experimental possibilities with smaller crystals. This approach requires new crystal preparation methods for specialized delivery systems. In this thesis, a technique for growing membrane protein micro-crystals using lipidic cubic phase crystallization and adding crystal seeds is presented. With this approach, a 2.3 Å resolution SFX structure of the reaction center was obtained. Detergent grown micro-crystals were used to validate a fixed target device, yielding a 3.3 Å structure from data collected using serial synchrotron X-ray crystallography. LCP grown micro-crystals were used in time resolved SFX studies, where structural movements of the co-factors in the protein on a sub-picosecond timescale after photon absorption were assessed. Further, time resolved SFX results show multi-photon absorption induces a thermally driven expansion of the protein structure surrounding the co-factors. These findings contribute to a sequence of studies that highlight the intricate interplay between light absorption and structural changes within the protein.