Artificial photosynthesis has recognised potential to produce green and sustainable fuels from earth-abundant resources such as water, carbon dioxide (CO2), and sunlight. In an artificial... Show moreArtificial photosynthesis has recognised potential to produce green and sustainable fuels from earth-abundant resources such as water, carbon dioxide (CO2), and sunlight. In an artificial photosynthetic system, two half-reactions, such as water oxidation and proton reduction or CO2 reduction, have to be combined. To achieve such a system, it is crucial to have: a) efficient light-harvesting by the photosensitiser, b) stable catalysts for the oxidation and the reduction reaction, c) unidirectional proton and electron transport between the oxidation and the reduction site, ideally by a recyclable electron relay, d) efficient charge separation, and e) a strong, photostable membrane that does not leak molecular components. In natural photosynthesis, these requirements are achieved altogether using compartmentalisation, which consists in embedding the key components of the system, i.e. for green plants the oxygen evolving complex, photosystem I and II, and the natural electron relays, around the lipid bilayer of the thylakoid membrane. The use of spherical lipid membranes (such as liposomes) as biological mimics of the thylakoid membrane is a promising approach to confine half-reactions, facilitate charge separation, and avoid charge recombination and other undesired side-reactions. In the research described in this thesis, it was attempted to realise a full artificial photosynthetic system based on liposomes and several of the key intermediate steps were achieved: 1) unidirectional electron transfer across a liposomal membrane from an electron donor encapsulated in the interior of the liposome to an electron acceptor located outside (Chapter 2), and 2) photocatalytic reduction of CO2 (Chapter 3) and of protons (Chapter 4) at the surface of liposomes. Special attention was paid in Chapter 2 and Chapter 5 to the question of the (photo)stability of the membrane and light-induced leakage. Show less
Ever since the structural data of biological macromolecules became available, there has been consistent struggle to relate this new information to the existing spectroscopy, activity and... Show moreEver since the structural data of biological macromolecules became available, there has been consistent struggle to relate this new information to the existing spectroscopy, activity and theoretical descriptions of these proteins and to understand the evolution and/or to predict the role of yet uncharacterized gene products in this light. The research presented in this thesis primarily deals with understanding the structure__function relationship of a newly discovered blue copper protein. The protein is derived from Streptomyces coelicolor and is called small laccase (SLAC). It utilizes four copper ions to catalyze the oxidation of substrate molecules concomitant with the reduction of oxygen to water. The catalytic cycle of this enzyme is studied using a variety of spectroscopic and kinetic methods in an attempt to improve our understanding of the internal operations which are critical to its functioning. The new results obtained are presented in this thesis. Show less
Photosynthesis in plants, algae and cyanobacteria releases oxygen and is referred to as oxygenic photosynthesis. Among all the photosynthetic reaction centers only PSII provides a redox potential... Show morePhotosynthesis in plants, algae and cyanobacteria releases oxygen and is referred to as oxygenic photosynthesis. Among all the photosynthetic reaction centers only PSII provides a redox potential sufficiently strong for water oxidation. While the electron flow in PSII is strictly asymmetric, in PSI different levels of bidirectionality indicate functional flexibility of this complex. Photo-CIDNP is non-Boltzmann nuclear magnetization caused by photochemical reactions and can be observed by NMR spectroscopy as strongly enhanced absorptive or emissive signals. In this thesis photo-CIDNP solid state NMR with selective isotope labeling is applied to get direct access to the heart of large PSII and PSI photosynthetic complexes in intact and isolated systems of Spirodela oligorrhiza and Synechocyctis. For the first time the direct observation of selective atoms within the heart of the PSII and PSI complexes by experiment s on entire plants and whole cells is reported. In this way the conservation of the electronic structure of the PSII electron donor at various levels of biological preparations have been addressed and the electron spin density (ρi) in the active cofactors of PSI has been constructed. In addition the functional heterogeneity of the PSI electron donor among different plant species was probed. Show less
Protein-protein interactions play an important role in all cellular processes such as signal transduction, electron transfer, gene regulation, transcription, and translation. Understanding these... Show moreProtein-protein interactions play an important role in all cellular processes such as signal transduction, electron transfer, gene regulation, transcription, and translation. Understanding these protein-protein interactions at the molecular level, is an important aim in structural biology. The protein interactions studied in this work are principally involved in the signal transduction and in electron transfer. These protein interactions belong to the family of transient dynamic interactions, meaning they associate and dissociate rapidly. The work presented in chapters two and three gives a detailed description of a study to determine the binding orientation of focal adhesion kinase derived peptide on the Src SH3 domain using paramagnetic NMR spectroscopy. The work presented in chapter four gives detailed information on the cloning, expression and purification of the focal adhesion kinase domain with the SH3 and S H2 binding sequences (32k) using a baculovirus expression system. The results of NMR characterization were presented for the complexes of different Src domains and the 32k. The work presented in the chapters five and six describes the NMR characterization of the interactions between several redox proteins. The aim of the work was to convert the transiently bound weak protein complexes into specific complexes Show less
The growing field of bio-electronics holds many promises with regard to the integration of various organic molecules onto printed circuit-boards. These include a decrease in the cost of production,... Show moreThe growing field of bio-electronics holds many promises with regard to the integration of various organic molecules onto printed circuit-boards. These include a decrease in the cost of production, an increased sensitivity and specificity to molecular detection from various solutions (i.e. blood) and ultra-miniaturization. However, numerous challenges still face such prospects, chief among which is the retention of biological activity of the adsorbed molecules. To circumvent the possible harmful effects of the bare surfaces, we have made use of self-assembled molecular films that not only shield the proteins (i.e. azurin) off surfaces, but also help establish a spatially-defined conductive path to electrodes. At the same time, the protein itself was engineered such that the active cavity is directly connected via such molecular __wires__. Our results may help in the adsorption of more complex enzymes into future molecular devices, that will retain their activity on the surfaces and are able to integrate into biosensors. Show less
The interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated... Show moreThe interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated cellular context. Recently, it was found that an intermediate state called encounter state, of a protein complex, exists briefly before a final protein complex is formed. In the encounter state, one protein is rolling over on the surface of its partner, searching for the optimal orientation. In my PhD thesis, a transient electron transfer complex formed between a heme protein and an iron-sulfur protein was found to be trapped in this intermediate state, existing as a pure encounter complex. Thus, characterization of this dynamic complex by nuclear magnetic resonance spectroscopy advances our understanding of the general mechanism of protein-protein interaction Show less
Photochemically induced dynamic nuclear polarization (photo-CIDNP) is non-Boltzmann nuclear magnetization which can be observed by magic angle spinning NMR spectroscopy as enhanced absorptive or... Show morePhotochemically induced dynamic nuclear polarization (photo-CIDNP) is non-Boltzmann nuclear magnetization which can be observed by magic angle spinning NMR spectroscopy as enhanced absorptive or emissive signals. In solids, photo-CIDNP has been observed since its discovery in 1994 in various photosynthetic reaction centers. In natural photosynthetic charge separation, electron-electron interactions are fine-tuned to lead to highly efficient electron transfer. Nanosecond laser-flash photo-CIDNP magic-angle spinning NMR allows for determination of the nuclear polarizations and hyperfine interactions with atomic selectivity and with a resolution of a few microseconds. The build-up of nuclear polarization in reaction centers of Rhodobacter sphaeroides is found to depend on the presence and lifetimes of the molecular triplet states of the donor and carotenoid. Time-resolved 13C photo-CIDNP MAS NMR spectroscopy is used to map the electronic structure of the donor. In the dark state, maximum electron density is localized in the center of the special pair. In contrast, in the light state, the maximum of the electron spin density is localized at the periphery of the two cofactors. The balance of electron spin density between the two bacteriochlorophyll cofactors is shifted in favor of the L branch of the protein by the ratio of 7:3. We show that the asymmetry is induced by both geometric differences between the two cofactors and non-covalent interactions with the protein. Show less
