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Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2020). = Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020, ATP synthase is an enzyme that catalyzes the synthesis of adenosine triphosphate (ATP) – the molecule which supplies energy for vital biochemical reactions in almost all living organisms. F-type ATP synthases are spread in bacteria cellular membranes, thylakoid membranes of chloroplasts and mitochondria inner membranes of eukaryotes. F-type ATP synthase is a membrane protein complex with a large non-membrane part F1 and a membrane part FO. Being so widely spread in different species, ATP synthases have conserved parts responsible for their major functions: ATP synthesis/hydrolysis and transmembrane ion transport in F1 and FO regions, respectively. At the same time their subunit organization and composition are highly variable, especially for mitochondrial F-type ATP synthase dimers in eukaryotes (four different types were found up to date). Bacterial ATP synthases are found in monomeric state as well as chloroplast ATP synthases, which are also predominantly monomers, however, there are protein pairs and oligomers of ATP synthases in chloroplasts according to the literature. At the moment the physiological role of such oligomers is discussed. One of the most important and unknown parts of ATP synthases – the membrane FO region, in particular, a rotary part (cn-ring), which consists of n copies of protomers (c1-subunits) that are assembled in a circle. Being coupled with the a-subunit, c-ring establishes a transmembrane ion transport, rotates γ-subunit and drives ATP synthesis. Despite the fact that ATP synthase was in a focus of interest for the last decades and resulted in a huge progress in its’ structural and functional studies, a number of key details remained unknown. High-resolution structures (better than 3 Å) were obtained for c-rings from bacterial and mitochondrial ATP synthases, however, the gap of the knowledge of a c-ring from chloroplasts was not filled. Another open question relates to molecular composition of the inner pore of c-rings. Literature data showed at least four different types of interaction interfaces of c-rings in a context of assembled ATP synthase dimers in mitochondria of eukaryotes. In all these cases, additional subunits interacting with a c-ring except a and γ subunits are also involved into dimerization FO/FO interface between two monomers of ATP synthase. However, in case of monomeric bacterial and chloroplast ATP synthases, no additional machinery, which could provide a similar stabilization of a c-ring, was found in FO part. The question of molecular composition of the inner pore of c-rings from different organisms, e.g. bacteria and chloroplasts, was not deeply discussed in literature. In the literature it was shown that pigments, e.g. chlorophyll a and beta carotene, are tightly connected to a c-ring from spinach chloroplasts and might be compounds of a lipid “plug” inside its’ inner pore, however, due to the absence of a c-ring structure it was not proven that the pigments were exactly inside the c-ring. In the present work the high-resolution structure (2.3 Å) of a c-ring of F-type ATP synthase from spinach chloroplasts was obtained. This is the first high-resolution structure of a c-ring from plant chloroplasts and the first subunits of any ATP synthase that were crystallized by in meso method. The present work fills the gap in knowledge of c-rings from plant chloroplasts and provides a complete c1/c1 interaction interface formed by a net of hydrogen bindings between amino acids of neighbor protomers. The role of the motifs G(A,S)xxxG(A,S) was theoretically predicted in literature to be key determinants of c-ring stoichiometry in different organisms. In my thesis, it is directly shown that the motifs G(A,S)xxxG(A,S) occurred to be only partially involved in a network of hydrogen bonds at the c1/c1 interface. It was also demonstrated that the complete network of hydrogen bonds includes the motifs G(A,S)xxxG(A,S) and other amino acids which determine the c1/c1 interface and, therefore, a stoichiometry of the c-ring.I found additional positive electron densities inside the c-ring. They were not associated with the c-subunits. I found similar densities during analysis of high-resolution structures of c-rings from different organisms, e.g. bacteria and mitochondria of eukaryotes. It was not previously discussed in the literature. The distances between polar/apolar interfaces were calculated and showed significant difference between inside the inner pore and outside of the c-ring. The hydrophobic length inside the c-ring was found to be ~1.4 times larger than in the membrane bilayer outside the c-ring. A similar difference (~1.2 – 1.4 times) was found for all c-rings with available high-resolution structures. Our hypothesis is that isoprenoid quinones inside the inner pore of c-rings may explain our and literature data. We suggest that these molecules are present in all c-rings (e.g. plastoquinone-9 (PQ-9) in a c-ring from spinach chloroplasts, menaquinones in bacteria, ubiquinones in mitochondria of eukaryotes). I tested the hypothesis by UV-Vis differential spectroscopy of the c-ring samples using a qualitative reaction of reducing ketones and aldehydes to alcohols by NaBH4, and showed the presence of the compounds with quinone group similar to trimethyl benzoquinone – a polar moiety of PQ-9. Thus, this presents, although indirect, arguments towards the hypothesis that isoprenoid quinones might be universal cofactors of ATP synthases, preventing ion leakage through a c-ring and stabilizing it. In addition, I studied the structure of ATP synthase. In the work, small-angle X-ray scattering (SAXS) of purified intact ATP synthase from spinach chloroplasts reconstituted into POPC/MSP1E3D1 nanodiscs was performed and showed the low-resolution structure that has a form-factor similar to that of V-shape dimers of mitochondrial ATP synthases. It rises questions of possible physiological role of such oligomers of chloroplast ATP synthases. Understanding of ATP synthase in details is extremely important. ATP synthase is a part of oxidative and photophosphorylation systems and relevant studies reveal hidden connections of ATP synthase in different biochemical processes. Dysfunctions of this enzyme lead to a number of severe diseases, e.g. as diabetes, severe metabolic and mitochondrial disorders, dysfunctions of tissues and organs, neurodegenerative and age-associated diseases. At the same time, ATP synthase, due to being significantly variable in details in organisms from distant lineages, might become a target for structure-based design of new antibacterial drugs. Thus, new insights on its’ structural and functional features are important for pharmacology and applied medicine, for development of different therapies and structure-based drug design as well as for fundamental biology. I believe that this work is contributing to better understanding of this molecular machine., Published by RWTH Aachen University, Aachen