Mass Spectrometry-Based Structural Analysis of Photosynthetic Protein Assemblies

This dissertation focuses on using mass spectrometry-based techniques to study photosynthetic protein assemblies. Photosynthesis is a process that converts light energy into chemical energy, the basis of most life on Earth. The two most crucial protein machineries involved in this process are reaction center and light harvesting complexes. They are usually giant protein complexes with different numbers of co-factors. In a more expanded sense, photosynthesis is not just about the utilization of solar energy, the regulation of light energy is also essential as excess light energy is detrimental to photosynthesis organisms. Again, protein assemblies play an indispensable role in this process. The knowledge of the structure and function as well as the molecular mechanism of those protein complexes are desired.Today, mass spectrometry is being widely used in proteomics studies. Its capabilities include but are not limited to the protein primary structure investigation. The development of MS-based footprinting, native MS and membrane protein MS detection platforms largely benefit the study of photosynthetic proteins. The MS-based footprinting technique can investigate protein conformational change upon its binding to other molecules or under the stimulus of pH change or other factors. Native MS can investigate the conformation and topology of protein complexes in a near-native environment where the non-covalent interactions are preserved. Membrane proteins are notoriously difficult to study. The development of MS-based membrane protein detection platforms largely benefits the study of photosynthesis, as reaction center and light-harvesting complexes are usually membrane proteins.In this dissertation, a variety of MS-based techniques were utilized to study reaction center proteins, light harvesting proteins and the proteins involved in the photoprotection process. We utilized top-down MS to study the components as well the primary structure of LH2 from a purple bacterium (Rb. sphaeroides), which reveals a new post-translational modification and mutation information. In addition, we developed a MS-based platform to footprint this LH2, investigating its topology in a lipid bilayer. The reaction center from another purple bacterium (B. viridis) was studied by both bottom-up and top-down MS and lots of unexpected mutations were identified. We also conducted a native MS study on this reaction center, and the capabilities of retaining the co-factors as well as its collisional cross section in the gas phase are discussed. Lastly, we study the orange carotenoid protein (OCP) and the fluorescence recovery protein, two major players in the non-photochemical quenching process in cyanobacteria. We utilized MS-based techniques to probe the conformation and structure of these two proteins and finally proposed a mechanism for non-photochemical quenching in cyanobacteri