Les métaux sont impliqués dans de nombreux processus biologiques essentiels pour le vivant. Ils interviennent au sein de métallo-enzymes sélectives et efficaces, qui catalysent des réactions chimiques dans des conditions douces. Les plus illustres sont les RiboNucléotide Réductases (RNR), essentiels à la synthèse de l'ADN, ou bien encore la Méthane MonoOxygènase (MMO) capable à partir du méthane de former le méthanol, molécule à fort potentiel énergétique. Ces métallo-enzymes fonctionnent au travers d'un site actif contenant deux fers. Récemment, un nouveau membre de cette famille a été isolé et présente un nouveau site actif hétérodinucléaire à fer et manganèse. Le potentiel chimique de ces enzymes commence juste a être caractérisé, mais les premières études suggèrent une réactivité semblable aux enzymes homodinucléaires à fer. Puisque le comportement de l'ion métallique dans les protéines n'est pas très différent de la chimie fondamentale du métal, l'étude de petits analogues synthétiques de site actif est particulièrement utile. Nous proposons la synthèse de complexes dinucléaires à Fe-Mn pour étudier la réactivité et les propriétés électroniques de ce nouveau site actif. Par une étude physicochimique approfondie et des études de réactivités, nous avons apporté une meilleure compréhension sur la réactivité de ce nouveau système enzymatique. / Nonheme enzymes possessing a dinuclear active site are performing many essential functions such as Ribonucleotide reductase (RNR) in DNA production and Methane oxygenation (MMO) to convert gas toxic methane in combustible methanol. While most of these enzymes have been shown to possess a diiron active site, new members of this protein family were recently isolated from bacteria and found to possess instead a heterodinuclear Fe-Mn active site. The chemical potential of the heterodinuclear metal site is just starting to be evaluated, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor. In recent years, the study of models based on simple dinuclear metal complexes has became an important tool for gaining insight into the biological functions of such bimetallic cores. The design of binucleating ligands capable of providing asymmetric dinuclear complexes is a subject of great interest. We propose to synthesize dinuclear Fe-Mn complexes to investigate the reactivity and the electronic properties of this new active site. By combining spectroscopic and electronical studies we have gain a better understanding on the reactivity of this new enzymatic system.

Diplomová práce se zabývá návrhem a sestavením nového spektrometru opticky detekované magnetické rezonance (ODMR) modifikací stávajícího spektrometru magnetického kruhového dichroismu (MCD) přivedením mikrovlnného ozařování. Je navrhnut nový držák vzorku umožnující osvětlení jak viditelným světlem, tak mikrovlnným zářením. Pro přivedení vlnění o nižších frekvencích je navržena anténa, určená k umístění pod vzorkem. Schopnosti celého systému jsou demonstrovány na sloučenínách kovových komplexů.

This diploma´s thesis deals with the determination of total antioxidant activity of red and white gooseberries and red, white and black currants. There are available information about small fruits, an overview of natural and synthetic antioxidants and a summary of the methods to determine the total antioxidant activity in the theoretical part. Frozen fruit samples were provided by Research and Breeding Institute of Pomology Holovousy Ltd. in the project QI111A141 – Research of new technologies in the cultivation of gooseberries and currants with a focus on quality and use of fetuses (Ministry of Agriculture). The total antioxidant activity of red and white gooseberries and currants was measured by spectrophotometric methods using free radicals ABTS•+ and DPPH and was expressed as a Trolox equivalent. The total antioxidant activity of black currants was measured by electron paramagnetic resonance using free radicals ABTS•+ and DPPH and expressed as a TEACABTS and a TEACDPPH. There were measured 8 species of red gooseberries, 6 species of white gooseberries, 12 species of red currants, 6 species of white currants and 8 species of black currants in this thesis. The highest values of total antioxidant activity showed species of red and white gooseberries Alan, Karmen, Pax and Invicta and species of red and white currants Rovada, Orion and Olin. Among black currants, species Ometa and Ruben had the highest values of total antioxidant activity.

This PhD. thesis is focused on application of different analytical techniques for determination of elemental composition of Moravian wine combined with analysis of selected polyphenol compounds. Two spectrometric techniques were used for elemental analysis of wine – Mass spectrometry and Optical emission spectrometry with inductively coupled plasma (ICP-MS and ICP-OES). Analysis of organic constituents of wine was evaluated by High performance liquid chromatograph with diode array detector (HPLC-DAD). Data from analysis of wine were used for construction of mathematical models for determining geographical origin of wine and for determining type of varietal wine. Data handling was performed by usage of multivariate statistical methods (cluster analysis, principal component analysis and canonical discriminant analysis). For the discrimination of wine by the geographical origin, two separate models for white and red wine were constructed. Effectiveness of discrimination of Moravian wine (with knowledge about origin) into 4 wine regions was 100 % successful for white wine and 96,43 % for red wine samples. Those models were used for cross validation test of unknown samples with result 95 % successfully classified samples of white wine and 82,15% of red wine. Besides that, mathematical model for discrimination and classification of varietal wine was made. Müller Thurgau, Riesling and Gruner Veltliner were discriminated by the usage of 8 elemental parameters. Discrimination success rate of known samples was evaluated by canonical discriminant analysis with 95,83% of correctly classified known samples and 70,83% of correctly classified unknown varietal wine (cross validation test). In addition to these part of research, impact of different grape production systems on elemental composition of wine and Vitis Vinifera was studied. The core of the study was to find the connections between the type of agriculture and chemical properties of wine and Vitis Vinifera. Two agriculture systems were assessed – Organic agriculture and Integrated pest management. . Results of this study showed that quantity and manner of pesticide application is very important factor affecting the final elemental composition of the grapevine and wine, especially the content of copper, zinc and manganese. That knowledge can be used in the future for creation of classification model of wine according to grape production system. For comparison of radical scavenging activity, electron paramagnetic resonance spectroscopy (EPR) with DPPH method was used. In the case of this study, the influence of vine growing under different farming systems on the total antioxidant activity of wine was found to be insignificant.

No description available.

The low-temperature binding of nitric oxide (NO) in the metal-organic framework MIL-100(Al) has been investigated by pulsed electron nuclear double resonance and hyperfine sublevel correlation spectroscopy. Three NO adsorption species have been identified. Among them, one species has been verified experimentally to bind directly to an 27Al atom and all its relevant 14N and 27Al hyperfine interaction parameters have been determined spectroscopically. Those parameters fit well to the calculated ones of a theoretical cluster model, which was derived by density functional theory (DFT) in the present work and describes the low temperature binding of NO to the regular coordinatively unsaturated Al3+ site of the MIL-100(Al) structure. As a result, the Lewis acidity of that site has been characterized using the NO molecule as an electron paramagnetic resonance active probe. The DFT derived wave function analysis revealed a bent end-on coordination of the NO molecule adsorbed at that site which is almost purely ionic and has a weak binding energy. The calculated flat potential energy surface of this species indicates the ability of the NO molecule to freely rotate at intermediate temperatures while it is still binding to the Al3+ site. For the other two NO adsorption species, no structural models could be derived, but one of them is indicated to be adsorbed at the organic part of the metal-organic framework. Hyperfine interactions with protons, weakly coupled to the observed NO adsorption species, have also been measured by pulsed electron paramagnetic resonance and found to be consistent with their attribution to protons of the MIL-100(Al) benzenetricarboxylate ligand molecules.

Sn2P2S6 is ferroelectric at room temperature. It is of technological interest in a variety of applications such as pyroelectric motion detectors and the photorefractive effect. Until now the defect structure and the light-induced charge transfer processes in this material have not been subject of a detailed investigation. The main part of this thesis describes and interprets data of electron paramagnetic resonance (EPR), optical absorption spectroscopy and their combination at 10 K to unravel the light-induced sensitization and charge transfer paths. In the photosensitized crystal at excitation with 1.5 eV a hole is transferred from a previously generated Sn3(plus) site to another inequivalent site of the ferroelectric phase. For higher excitation energies another hole present as Fe3(plus) is transferred to S2- creating S-. Optical absorption spectroscopy at room temperature indicates the validity of this model for evelated temperature below the Curie-temperature as well. It is consistent with both our interpretation of EPR spectra and the observation of photoinduced persistent conductivity that electronic bipolarons are the negative charge carriers. An additional chapter interprets a manifestation of pyroelectric luminescence already reported in other pyroelectric materials in terms of an internal Poole-Frenkel-effect induced by the pyroelectric field under changing temperature. The numerical simulation based on published material parameters is in good agreement with the experimental data for both heating and cooling.

The cytoskeletal protein vinculin plays a key role in the control of cell-cell or cell-matrix adhesions. It is involved in the assembly and disassembly of focal adhesions and affects their mechanical stability. While many facts highlight the importance and significance of vinculin for vital processes, its precise role in the regulation of cell adhesions is still only partially understood. Various EPR methods are used in this work in order to study the vinculin tail (Vt) domain in an aqueous buffer solution and its structural changes induced by F-actin and acidic phospholipids. EPR results in combination with a rotamer library approach (RLA), MD simulation and other computational methods allowed the construction of molecular models of Vt and dimeric Vt in the presence and absence of its binding partners. Furthermore, X-band orientation selective DEER measurements were applied on a Vt double mutant. It could be shown that the determination of the mutual orientation of protein bound spin labels is possible at X-band frequencies, if the orientation correlation of the spin label pair is strong. The method established here can be used to determine valuable information about proteins and nucleic acids, expanding the virtue of DEER spectroscopy as a tool for structure determination.

Understanding the conformational and dynamic changes of biomacromolecular complexes in different states, such as the membrane protein photoreceptor-transducer complex NpSRII/NpHtrII, is a key step to gaining insight into the functional mechanism of these important classes of protein complexes, since ~30 % of the human proteome are membrane proteins, yet they are largely underrepresented in terms of structural information with <1 % of all structures in the protein data bank. Hence for the development of methods suitable to study the conformation and dynamics of such complexes there is a strong demand and a vast potential field of applications. Here we combined method development at the interface between biomolecular simulations and model-based analysis of EPR- and fluorescence spectroscopic data with application studies using state-of-the-art spectroscopic techniques in conjunction with site-directed spin- or fluorescence labeling. In an initial benchmark study on the rigid globular protein complex Rpo4/7, we compared experimental inter fluorescence label distances or spin label distance distributions to a variety of predicted inter label distances based on molecular dynamics simulations, Monte Carlo sampling and a discrete rotamer library analysis. We found that while for the molecular dynamics simulations with explicit solvent considerable sampling challenges have to be overcome to reproduce the experimentally observed inter label distance distributions, the Monte Carlo sampling performed well when compared to the experimental data and was computationally less demanding. Significantly more efficient and equally accurate for our examples was the so-called rotamer library analysis available for the spin labels since it relies on a pre-calculated set of rotational isomers. In general, predictions for the mean distances were in agreement within the error margins while distribution shapes were more challenging to reproduce. Overall this study shows a positive evaluation for the assessed tools and the developed simulation protocols as well as their potential applications. Using the combination of EPR and fluorescence spectroscopy for distance determination we studied the structural influence of RNA binding on Rpo4/7, and showed that the protein complex stays conformationally rigid and thereby serves as a guiding rail for the nascent RNA chain that leaves the RNA polymerase along the Rpo4/7 RNA binding interface. To enhance the interpretation of experimentally determined changes of conformation and dynamics in protein complexes and to discuss the observed changes in terms of structural information, we built models of the two transcription factors TFE and the Spt4/5 complex, as well as of Argonaute, a 713 amino acid four-domain protein nuclease from Methanocaldococcus jannaschii. These structural models not only allowed a more accurate planning of fluorescence or EPR labeling experiments, but also the models enabled the discussion of the experimental data in structural terms. Based on such an initial structure further computational analysis techniques may be applied to identify putative structural changes or dynamic modes. This was shown for the histidine transporter HisQMP2, where we combined normal mode analysis to model protein flexibility with the rotamer library analysis to screen for possible conformational changes in comparison to experimental inter spin distance data. The most prominent agreement with one mode led to a working hypothesis of a conformational change and provides the basis for validation in future experiments. Due to the inherent synergy effects, we applied a combined experimental and simulation approach for the EPR-based distance determination in the globular DNA-binding protein LexA to probe conformation and dynamics of the N-terminal DNA-binding domains with respect to the C-terminal domains within the LexA homodimer. While the C-terminal dimerization domains exhibit a well-defined conformation that proved to be independent of DNA-binding, large-scale changes in conformation and dynamics were detected for the N-terminal domains. They were only found in a defined conformation when bound to DNA while in its absence a large rotational freedom of the entire N-terminal domains contributed to the conformational ensemble. Combined with a biochemical characterization of the autocatalytic cleavage of LexA, our data explains how LexA induces the SOS response after DNA damage or under latent antibiotic stress. We further studied the membrane photoreceptor-transducer complex NpSRII/NpHtrII that governs the light-dependent swimming behavior in Natronomonas pharaonis by a two-component signaling system. This system comprises extraordinary features of sensitivity, signal amplification, integration and transducer cooperativity, yet the molecular details of these features are poorly understood, as is signal propagation itself. By combining time-resolved cw EPR spectroscopy of NpSRII/NpHtrII variants spin labeled in the HAMP1 domain with time-resolved optical absorbance spectroscopy to report on the receptor signaling state, we found a tight kinetic coupling of receptor and transducer during the relaxation back to the ground state and hence a prolonged activation period, that with ~500 - ~700 ms is sufficiently long to cause phosphorylation bursts of the cognate kinase CheA. This explains signal amplification already on the level of the NpSRII/NpHtrII dimers. We further determined the transient difference spectra from the time-resolved EPR data that show local differences in dynamics and steric restrictions upon light-activation. Comparing these experimentally observed differences to predictions confirms the assumed two-state structural model and shows this transition between the two states for a single HAMP domain in a light-dependent manner. Additionally, our approach integrates a dynamic view into the model, since the two states are shown to exhibit different local dynamics in a fashion described previously as a competing model for signaling by dynamic differences based on biochemical studies. Here we show unification of the two models into one congruent description encompassing a transition between the two previously suggested states by concerted structural and dynamic changes. In an independent analysis using all-atom and coarse grained molecular dynamics of the NpSRII/NpHtrII complex in the minimal unit that can exert kinase control, the trimer of receptor-transducer dimers, we revealed a distinct dynamical pattern encoded in the primary sequence of the coiled-coil heptad-repeats. Upon receptor activation, these segments alter their dynamics in a concerted fashion with regions such as HAMP1 and the adaptation region becoming more compact, while HAMP2 and the tip become more dynamic, leading to dynamic and to limited structural changes at the CheA-kinase binding sites. Together with an extensive validation against experimental data, these findings suggest the altered dynamics as the mechanism for signal propagation along the extended coiled-coil structure of NpHtrII. This working model, that explains the current body of experimental data, allows for further refinement by all-atom molecular dynamics and provides a basis to devise future experiments for validation. The presented studies outline the versatile methodology of combined experimental and simulation approaches to analyze the conformation and dynamics of biomacromolecules including membrane protein complexes.

The photosensitive unit triggering the negative phototaxis in the haloarchaeum Natronomonas pharaonis consists of the receptor sensory rhodopsin II (NpSRII) and its cognate transducer (NpHtrII) in a 2:2 stoichiometry. Upon light excitation, a structural rearrangement in the receptor initiates a displacement/rotation of the transducer helix TM2, which can be considered as starting event for the signal transduction. This signal is further transmitted to the cytoplasmic signaling domain through the signal transduction unit comprising two HAMP domains.Structural information already exists for the transmembrane region of this complex (crystal structure) as well as for the rod shaped cytoplasmic part of NpHtrII due to its high homologies with chemoreceptors. Moreover, the solution NMR structure of the isolated HAMP domain from A. fulgidus recently obtained shows a homodimeric, four-helical, parallel coiled-coil with an unusual interhelical packing, that is thought to propagate a signal by virtue of concerted helix rotations. Here, an electron paramagnetic resonance (EPR) investigation of site-directed spin labeled transducers in the NpSRII/NpHtrII complex has been carried out for structural and functional elucidation of the N. pharaonis HAMP. For this purpose, cw as well as pulse EPR techniques have been used in terms of mobility, accessibility and intra-transducer dimer distance analyses. Conformational changes induced by environmental inputs, namely salt, temperature and pH, give insight into the two-state equilibrium existing between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation of this linker region.