In dieser Arbeit werden die Synthese eines 286-Reste-langen Proteins, der γ-Untereinheit der ATP-Synthase, seine Rückfaltung und Rekonstitution zum aktiven Proteinkomplex gezeigt.

We have studied the inhibitory effect of natural and structurally modified polyphenols on Escherichia coli ATP synthase to test (I) if the beneficial dietary effects of polyphenols are related to their inhibitory actions on ATP synthase, (II) if inhibitory effects of polyphenolic compound could be augmented through structural modifications, and (III) if they can act as antimicrobial agent through their actions on ATP synthesis. X-ray crystal structures of polyphenol binding sites suggested that polyphenols bind at a distinct polyphenol binding pocket, at the interface of α,β,γ-subunits. We found that both natural and modified polyphenols inhibit E. coli ATP synthase to varying degrees and structural modifications resulted in augmented inhibition. Inhibition was reversible in all cases. Both natural and modulated compounds inhibited E. coli cell growth to varying degrees. We conclude that dietary benefits of polyphenols may be in part due to the inhibition of ATP synthase.

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ATP Synthase is the fundamental means of cellular energy production in animals, plants, and almost all microorganisms. In order to understand the mechanism of ATP catalysis, critical amino acid residues involved in Pi binding have to be identified. The αVISIT-DG sequence at the interface of α/β subunits that contains residues from 345-351 is highly conserved and αAsp-350 has been chosen because of its negative charge side chain and its close proximity (~2.8 Å) to the known phosphate binding residue αArg-376. The mutant's αD350R, αD350Q, αD350A, αR376A/D, and αG351R/A/D were generated by site directed mutagenesis and several biochemical assays were performed on them to understand the role played by the amino acid residues in Pi binding. Biochemical results suggest that αD350 may be involved in catalysis of ATP synthase and play an important role in Pi binding, whereas αG351 may be involved only in the structural integrity.

ATP synthase is an essential protein complex located in the mitochondrial inner membrane, which synthesize ATP by coupling to a rotary proton transport across the membrane at the expense of the electrochemical proton gradient created by the electron transport chain. This reaction guarantees the supply of energy to biological processes in a cell. When mitochondria get deenergized, i.e. the protomotive force across the mitochondrial inner membrane collapses, the ATP synthase switches from ATP synthesis to hydrolysis. This hydrolytic activity is then immediately prevented by a natural soluble mitochondrial ATPase inhibitor, IF1. This efficient reversible inhibition system protects cells from wasting energy. In yeast, IF1 is a small protein consisting of 63 amino acids. It binds to one of the three (αβ) catalytic interfaces of ATP synthase and thereby blocks the rotary catalysis. Although the crystal structure of the dead-end IF1 inhibited F1-ATPase complex has been resolved, IF1 initial binding and locking to ATPase still remain unclear events at the molecular level.During my thesis, we have been interested in the dynamic mechanism of ATPase inhibition by IF1. By means of analyses of published structures and protein sequence alignment, we selected numerous residues located in different regions of Saccharomyces cerevisiae ATP synthase α, β subunits, which might potentially paticipate in IF1 binding process. Using site-directed mutagenesis combined with kinetic experiments, we studied the effect of mutations of the selected candidates on the rate and extent of ATPase inhibition by IF1. In this way we identified residues or motifs in ATP synthase α, β subunits involved in IF1 recognition and/or locking steps, which allows complementing structural studies and drawing an outline of IF1 binding.

Rare diseases are a heterogeneous group of disorders. Knowledge of their molecular basis is poor and till recently there were no appropriate methodical approaches due to a limited number of patients. Novel genomic techniques, especially the DNA array technology and the next generation sequencing emerging in last few years, enabled studies of these diseases even in small families and sporadic cases. This PhD thesis focuses on application of novel genomic techniques in studies of rare inherited diseases. It describes a use of DNA array technology in linkage analysis, analysis of differential gene expression, analysis of copy number variations and homozygous mapping, and a use of next generation sequencing technology. Combination of these methods was used for identification of molecular basis of adult neuronal ceroid lipofuscinosis, Rotor syndrome, isolated defect of ATP synthase and mucopolysaccharidosis type IIIC.

In this thesis, phylogenomic and comparative structural analyses of several widespread energy converting enzymes were performed. The focus was on the major subfamilies of the enzymes that process nucleoside triphosphates (ATP and GTP) and on some key enzymes of the electron transfer chains. First, we analyzed the P-loop GTPases, RadA/RecA recombinases, chaperone GroEL, branched-chain α-ketoacid dehydrogenase kinases, chaperone Hsc70, actins, and membrane pyrophosphatases. In the each inspected family we could identify (1) members which were potassium-dependent and/or contained K+ ions in the active site, and (2) potassium-independent enzymes with lysine or arginine residues as catalytic groups that occupy the positions of potassium ions in the homologous, K+-dependent enzymes. Based on the results of our analyses, we suggest that the appearance of the K+-binding sites could precede in evolution the recruitment of positively charged residues (lysine or arginine "fingers") with the latter providing more possibilities to control the enzyme reactions. Second, we have described the distinctive features of a phylogenetically separated subfamily of rotary membrane ATPases which we named N-ATPases. The N-ATPases have a specific operon organization with two additional subunits, absent in other rotary ATPases, and a complete set of Na+-binding ligands in the membrane c-subunits. We made a prediction, which was later confirmed, that these enzymes are capable of Na+ translocation across the membrane and may confer salt tolerance on marine prokaryotes. Third, phylogenomic analysis of the cytochrome bc complexes suggests that these enzyme complexes initially emerged within the bacteria and were then transferred to archaea via lateral gene transfer on several independent occasions. Our analysis indicates that the ancestral form of the cytochrome bc complex was a b6f-type complex; the fusion of the cytochrome b6 and the subunit IV to a "long" cytochrome b of the cytochrome bc1 complexes could have happened in different lineages independently. Fourth, our phylogenomic and comparative structural analyses of the cytochrome bc1 complex and of cytochrome c allowed us to trace how these enzymes became involved in triggering of apoptosis in Metazoa. We could trace the emergence of a specific cardiolipin-binding site within the cytochrome bc complex and the evolution of structural traits that account for the involvement of the cytochrome c as a trigger of apoptosis in vertebrates.

F₁-ATPase, the sector of ATP synthase where the synthesis of cellular ATP occurs, is a rotary molecular motor in its own right. Driven by ATP hydrolysis, direct observation of the rotation of the central axis within single molecules of F₁ is possible. Operating at close to 100% efficiency, F₁ from thermophilic Bacillus has been shown to produce ~40pN˙nm of torque during rotation. This thesis details the groundwork required for the direct measurement of the torque produced by F₁ using a rotary angle clamp, an optical trapping system specifically designed for application to rotary molecular motors. Proof-of-concept experiments will be presented thereby demonstrating the ability to directly manipulate single F₁ molecules from Escherichia coli and yeast mitochondria (Saccharomyces cerevisiae), along with activation of F₁ out of its inhibited state by the application of external torque. Despite in-depth knowledge of the rotary mechanism of F₁ from thermophilic Bacillus, the rotation of F₁ from Escherichia coli is relatively poorly understood. A detailed mechanical characterization of E.coli F₁ will be presented here, with particular attention to the ground states within the catalytic cycle, notably the ATP-binding state, the catalytic state and the inhibited state. The fundamental mechanism of E.coli F₁ appears to depart little from that of F₁ from thermophilic Bacillus, although, at room temperature, chemical processes occur faster within the E.coli enzyme, in line with considerations regarding the physiological conditions of the different species. Also presented here is the verification of the rotary nature of yeast mitochondrial F₁. The torque produced by F₁ from thermophilic Bacillus, E.coli and yeast mitochondria is the same, within experimental error, despite their diverse evolutionary and environmental origins.

Rare diseases represent a clinically and genetically heterogeneous group of diseases affecting various organs and presenting at different ages. Identification and functional characterization of genetic defects causing individual rare diseases represent unique opportunity to understand biological functions of human genes and gene products as well as to basic pathogenetic mechanisms of individual diseases. This knowledge is prerequisite for their effective diagnosis, specific treatment and prevention and it also opens up an avenue for better understanding of complex diseases. My thesis documents basic conceptual and methodological developments of biochemical genetics, functional cloning, genetic mapping, positional cloning, DNA microarrays and genomic sequencing, which have provided a universal framework for effective characterization of the genetic architecture of almost all human diseases. This conceptual and technological developments are demonstrated on several cases of rare genetic diseases - adenylosuccinate lyase deficiency, mucopolysacharidosis type IIIC, Rotor syndrome, deficiency of ATP synthase, neuronal ceroid lipofuscinosis, GAPO syndrome and X -linked restrictive cardiomyopathy, which genetic and molecular basis I have helped to elucidate.