Techn. Universiẗat, Diss., 2001-- Berlin.

Techn. University, Diss., 2003--Berlin.

Techn. Universiẗat, Diss., 2003--Berlin.

University, Diss., 2004--Jena.

This qualification work is trying to shed a little bit more light on some proteins present in higher plants, which structure and function in photosynthetic reaction remain unclear. In particular it treats proteins of photosystem II, called PsbR, PsbW and PsbX that are responsible for photosynthetic reaction optimization. This thesis contains data about proteins acquisition and their sequences elucidation.

abstract: Photosystem II (PSII) is a large protein-cofactor complex. The first step in photosynthesis involves the harvesting of light energy from the sun by the antenna (made of pigments) of the PSII trans-membrane complex. The harvested excitation energy is transferred from the antenna complex to the reaction center of the PSII, which leads to a light-driven charge separation event, from water to plastoquinone. This phenomenal process has been producing the oxygen that maintains the oxygenic environment of our planet for the past 2.5 billion years. The oxygen molecule formation involves the light-driven extraction of 4 electrons and protons from two water molecules through a multistep reaction, in which the Oxygen Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4. Unraveling the water-splitting mechanism remains as a grant challenge in the field of photosynthesis research. This requires the development of an entirely new capability, the ability to produce molecular movies. This dissertation advances a novel technique, Serial Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved molecular movies may be attained. The ultimate goal is to make a “molecular movie” that reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX) experiments and the uniquely enabling features of X-ray Free-Electron Laser (XFEL) for the study of biological processes. This thesis presents the development of SFX techniques, including development of new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL experiment) with the goal of solving the X-ray structures in different transition states. ii The research comprises significant advancements to XFEL software packages (e.g., Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the data acquired successfully. This research demonstrates that with manual optimizations, the evaluation success rate was enhanced to 40-50%. These improvements have enabled TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This breakthrough demonstrated the first indication of conformational changes between the ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical predictions. The power of the TR-SFX technique was further demonstrated with proof-of principle experiments on Photoactive Yellow Protein (PYP) micro-crystals that high temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved. In summary, this dissertation research heralds the development of the TR-SFX technique, protocols, and associated data analysis methods that will usher into practice a new era in structural biology for the recording of ‘molecular movies’ of any biomolecular process. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

abstract: Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule. This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2018

abstract: A vast amount of energy emanates from the sun, and at the distance of Earth, approximately 172,500 TW reaches the atmosphere. Of that, 80,600 TW reaches the surface with 15,600 TW falling on land. Photosynthesis converts 156 TW in the form of biomass, which represents all food/fuel for the biosphere with about 20 TW of the total product used by humans. Additionally, our society uses approximately 20 more TW of energy from ancient photosynthetic products i.e. fossil fuels. In order to mitigate climate problems, the carbon dioxide must be removed from the human energy usage by replacement or recycling as an energy carrier. Proposals have been made to process biomass into biofuels; this work demonstrates that current efficiencies of natural photosynthesis are inadequate for this purpose, the effects of fossil fuel replacement with biofuels is ecologically irresponsible, and new technologies are required to operate at sufficient efficiencies to utilize artificial solar-to-fuels systems. Herein a hybrid bioderived self-assembling hydrogen-evolving nanoparticle consisting of photosystem I (PSI) and platinum nanoclusters is demonstrated to operate with an overall efficiency of 6%, which exceeds that of land plants by more than an order of magnitude. The system was limited by the rate of electron donation to photooxidized PSI. Further work investigated the interactions of natural donor acceptor pairs of cytochrome c6 and PSI for the thermophilic cyanobacteria Thermosynechococcus elogantus BP1 and the red alga Galderia sulphuraria. The cyanobacterial system is typified by collisional control while the algal system demonstrates a population of prebound PSI-cytochrome c6 complexes with faster electron transfer rates. Combining the stability of cyanobacterial PSI and kinetics of the algal PSI:cytochrome would result in more efficient solar-to-fuel conversion. A second priority is the replacement of platinum with chemically abundant catalysts. In this work, protein scaffolds are employed using host-guest strategies to increase the stability of proton reduction catalysts and enhance the turnover number without the oxygen sensitivity of hydrogenases. Finally, design of unnatural electron transfer proteins are explored and may introduce a bioorthogonal method of introducing alternative electron transfer pathways in vitro or in vivo in the case of engineered photosynthetic organisms. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2014

Die pflanzliche Light-harvesting complex-Proteinfamilie besteht aus Proteinen mit vielfältigen Funktionen. Dabei ist die Funktion der Light-harvesting-like 3-Proteine (LIL3) sowie der One-helix-Proteine (OHPs) weitestgehend unbekannt. Im Rahmen dieser Arbeit wurde gezeigt, dass LIL3 nicht nur mit der Geranylgeranyl-Reduktase (CHLP), sondern auch mit der Protochlorophyllid-Oxidoreduktase (POR) interagiert. Sowohl CHLP als auch POR werden über die Interaktion zu LIL3 an die Thylakoidmembran gebunden und dadurch stabilisiert. Beide Enzyme liefern die direkten Vorstufen für den von der Chlorophyll-Synthase (CHLG) katalysierten finalen Chlorophyll-Syntheseschritt. Neben der Bestätigung der bereits früher gezeigten Chlorophyllbindung von LIL3 konnte eine Affinität zu den späten Intermediaten der Chlorophyllbiosynthese Proto IX, MgP, MgPMME und Pchlid nachgewiesen werden. Die größte Affinität bestand dabei gegenüber dem Substrat von POR, Pchlid. Basierend auf diesen Erkenntnissen wird LIL3 als Regulator der späten Chlorophyllbiosynthese-Schritte vorgeschlagen: LIL3 transportiert Substrate zwischen den Enzymen und ermöglicht durch die Bindung von CHLP und POR die Synthese der Chlorophyll-Edukte in räumlicher Nähe. Dadurch wird die Versorgung von CHLG mit dessen Edukten favorisiert. Beide OHP-Varianten (OHP1/2) bilden ausschließlich Heterodimere und binden Chlorophyll sowie Carotinoide im Verhältnis 3:1. Die Pigmentbindung basiert auf den konservierten Aminosäuren im Chlorophyllbindemotiv. An das OHP1-OHP2-Dimer bindet der PSII-Assemblierungsfaktor HCF244 und wird dadurch an der Membran verankert. HCF244 stabilisiert das OHP-Heterodimer und beide OHPs stabilisieren sich gegenseitig. Der heterotrimere OHP1-OHP2-HCF244-Komplex ist für die D1-Synthese wesentlich. Es wird vermutet, dass die OHPs an der co-translationalen Beladung von (p)D1 mit Pigmenten beteiligt sind sowie frühe Assemblierungsintermediate von PSII vor überschüssiger Anregungsenergie schützen. / The plant light-harvesting complex protein family comprises different members with a variety of functions. However, the function of the light-harvesting-like 3 proteins (LIL3) as well as the one-helix proteins (OHPs) is largely unknown. In this thesis, an interaction of LIL3 not only with geranylgeranyl-reductase (CHLP), but also with protochlorophyllide-oxidoreductase (POR) could be established. LIL3 tethers CHLP and POR to the thylakoid membrane, thereby conferring stability to both enzymes. Both CHLP and POR are synthesizing the direct chlorophyll precursors which are combined to chlorophyll by the subsequent chlorophyll synthase (CHLG). In addition to the chlorophyll binding ability of LIL3 reported earlier, an affinity of LIL3 towards the chlorophyll biosynthesis intermediates Proto IX, MgP, MgPMME, and Pchlide could be shown. Interestingly, the highest affinity of LIL3 was exerted towards Pchlide which is the substrate of POR. Therefore, LIL3 is postulated to shuffle the intermediates between enzymes and brings CHLP and POR in close proximity, which may help to supply CHLG with its substrates. Regarding the function of the OHPs an exclusive heterodimer formation of both the OHP1 and OHP2 variants could be shown. The OHP1-OHP2-heterodimer is able to bind chlorophyll and carotenoids in an approximate 3:1 ratio and pigment binding depends on dimer formation as well as the presence of the conserved amino acids in the chlorophyll binding motif. The PSII-assembly factor HCF244 is anchored to the thylakoid membrane by binding to both OHPs, thereby stabilizing the OHP-heterodimer. The heterotrimeric OHP1-OHP2-HCF244-complex is essential for D1 biosynthesis, although the exact molecular function of HCF244 is still unknown. It is suggested that the OHP-dimer is responsible for co-translational loading of (p)D1 with pigments as well as photoprotection of early PSII assembly intermediates.

Background: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. Results: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. Conclusion: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.