Physikalische Chemie
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Item Structure and flexibility of copper‐modified DNA G‐quadruplexes investigated by 19F ENDOR experiments at 34 GHz(2023-08-21) Schumann, Simon L.; Kotnig, Simon; Kutin, Yury; Drosou, Maria; Stratmann, Lukas M.; Streltsova, Yana; Schnegg, Alexander; Pantazis, Dimitrios A.; Clever, Guido H.; Kasanmascheff, MügeDNA G-quadruplexes (GQs) are of great interest due to their involvement in crucial biological processes such as telomerase maintenance and gene expression. Furthermore, they are reported as catalytically active DNAzymes and building blocks in bio-nanotechnology. GQs exhibit remarkable structural diversity and conformational heterogeneity, necessitating precise and reliable tools to unravel their structure-function relationships. Here, we present insights into the structure and conformational flexibility of a unimolecular GQ with high spatial resolution via electron-nuclear double resonance (ENDOR) experiments combined with Cu(II) and fluorine labeling. These findings showcase the successful application of the 19F-ENDOR methodology at 34 GHz, overcoming the limitations posed by the complexity and scarcity of higher-frequency spectrometers. Importantly, our approach retains both sensitivity and orientational resolution. This integrated study not only enhances our understanding of GQs but also expands the methodological toolbox for studying other macromolecules.Item Alcohol‐induced conformation changes and thermodynamic signatures in the binding of polyphenols to proline‐rich salivary proteins(2023-09-11) Jahmidi-Azizi, Nisrine; Oliva, Rosario; Winter, RolandThe first contact of polyphenols (tannins) with the human body occurs in the mouth, where they are known to interact with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for the development of astringency, but affect also various other biochemical processes. Employing thermodynamic measurements, fluorescence and CD spectroscopy, we investigated the binding process of the prototypical polyphenol ellagic acid (EA) to different IB-PRPs and BSA, also in the presence of ethanol, which is known to influence tannin–protein interactions. Binding of EA to BSA and the small peptide IB7-14 is weak, but very strong to IB9-37. The differences in binding strength and stoichiometry are due to differences in the binding motifs, which also lead to differences in the thermodynamic signatures of the binding process. EA binding to BSA is enthalpy-driven, whereas binding to both IB7-14 and IB9-37 is entropy-driven. The presence of 10 vol.% EtOH, as present in wines, increases the binding constant of EA with BSA and IB7-14 drastically, but not that with IB9-37; however, it changes the binding stoichiometry. These differences can be attributed to the effect of EtOH on the conformation dynamics of the proteins and to changes in hydration properties in alcoholic solution.Item Bacterial model membranes under the harsh subsurface conditions of Mars(2023-10-17) Tortorella, Attila; Oliva, Rosario; Giancola, Concetta; Petraccone, Luigi; Winter, RolandBiomembranes are a key component of all living systems. Most research on membranes is restricted to ambient physiological conditions. However, the influence of extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments, is less well understood. The deep subsurface of Mars is thought to harbour high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments. Here, we investigated the combined effects of high concentrations of Mars-relevant salts, including sodium and magnesium perchlorate and sulphate, and high hydrostatic pressure on the stability, structure, and function of a bacterial model membrane. To this end, several biophysical techniques have been employed, including calorimetry, fluorescence and CD spectroscopy, confocal microscopy, and small-angle X-ray scattering. We demonstrate that sulphate and perchlorate salts affect the properties of the membrane differently, depending on the counterion present (Na+vs. Mg2+). We found that the perchlorates, which are believed to be abundant salts in the Martian environment, induce a more hydrated and less ordered membrane, strongly favouring the physiologically relevant fluid-like phase of the membrane even under high-pressure stress. Moreover, we show that the activity of the phospholipase A2 is strongly modulated by both high pressure and salt. Compellingly, in the presence of the chaotropic perchlorate, the enzymatic reaction proceeded at a reasonable rate even in the presence of condensing Mg2+ and at high pressure, suggesting that bacterial membranes could still persist when challenged to function in such a highly stressed Martian environment.Item High pressure treatment promotes the deteriorating effect of cationic antimicrobial peptides on bacterial membranes(2023-03-29) Kriegler, Simon; Jaworek, Michel W.; Oliva, Rosario; Winter, RolandThe helical structure that cationic antimicrobial peptides (cAMPs) adopt upon interaction with membranes is key to their activity. We show that a high hydrostatic pressure not only increases the propensity of cAMPs to adopt a helical conformation in the presence of bacterial lipid bilayer membranes, but also in bulk solution, and the effect on bacterial membranes persists even up to 10 kbar. Therefore, high-pressure treatment could boost cAMP activity in high-pressure food processing to extend the shelf-life of food.Item Advanced EPR spectroscopic investigation of iron-sulfur clusters along the hydrogen evolution pathway(2023) Heghmanns, Melanie; Kasanmascheff, Müge; Happe, ThomasIron-sulfur (FeS) clusters are essential cofactors found in all living organisms. Acting as versatile electron carriers they are indispensable for life-sustaining processes, contributing to respiration, nitrogen fixation, and hydrogen production. Several proteins containing single and multiple FeS clusters are involved in the pathway to hydrogen evolution. Electron paramagnetic resonance (EPR) spectroscopy was used to characterize their FeS clusters providing valuable information about their magnetic properties, structural features, redox states, and biological function. Understanding and tuning the complex interplay of these proteins and their clusters is required for efficient biotechnological H2 production to sustainably meet the requirements of the world's increasing energy demand. We established pulsed EPR monitored redox potentiometry, performed at higher frequencies than usual, for determining the midpoint potentials of ferredoxins (Fdxs) and variants. Exchanging a single amino acid residue in CrFdx1 fine-tuned the midpoint potential of its [2Fe2S] cluster. Moreover, the characterization of fully maturated and more complex [FeFe]-hydrogenases harboring multiple distinct FeS clusters is addressed. One of the main drawbacks of efficient hydrogen production is the oxygen sensitivity of the H-cluster. A newly discovered oxygen-protection mechanism in the [FeFe]-hydrogenase from C. beijerinckii led to a comprehensive EPR spectroscopic study, characterizing not only the H-cluster states but also discovering a new radical R•ox. Advanced spectroscopic methods explored its origin and function. Eventually, the spectral features of the complex [FeFe]-hydrogenase CpI from C. pasteurianum were revisited and revealed exchange interactions between the H-cluster and the neighbored [4Fe4S] cluster. Variants showed a change in the strength of the exchange coupling and were explored to investigate its effect on the biological function. The studies presented in this thesis, including the fine-tuning of the midpoint potential of Fdxs, the discovery and characterization of an unusual radical signal, and the investigation of exchange coupling interactions in [FeFe]-hydrogenases, shed light on the magnetic properties and functional roles of FeS clusters in essential electron transfer processes and hydrogen evolution pathways.Item Structure and dynamics studies of the enzymes hCAII and GlpG via NMR spectroscopy(2024) Kotschy, Julia; Linser, Rasmus; Rauh, DanielLösungs- und Festkörper-NMR-Spektroskopie eignen sich gut, um Moleküle wie Proteine mit atomarer Auflösung zu charakterisieren. Hier wurden insbesondere strukturelle und dynamische Änderungen durch die Interaktion mit Inhibitoren, Strategien zur NMR-Probenoptimierung und verschiedene Methoden zur Zuordnung von NMR-Peaks eruiert. Lösungs- und Festkörper-NMR-Experimente wurden entwickelt, um die Flexibilität von SBR in hCAII-gebundenem Zustand zu messen. Das Enzym hCAII reguliert osmotische Prozesse in humanen Zellen und ist deshalb ein Zielprotein für Arzneimittel. Die entwickelte Methode zeigte, dass sich der Ligand trotz hoher Affinität in der Bindungstasche bewegt. Dies könnte von Interesse für die Wirkstoffentwicklung sein. Des Weiteren wurden sogenannte „time-shared“ 3D Lösungs- und Festkörper-NMR-Experimente für die Zuordnung von Methyl-Gruppen durch die Korrelation von Amid- und Methyl-Protonen in räumlicher Nähe zueinander genutzt. Intramembranproteasen der Rhomboid-ähnlichen Familie sind an Prozessen beteiligt, die mit Krebs- und neurodegenerativen Erkrankungen[1] assoziiert sind. Die Intramembranprotease GlpG aus E. coli wurde als Vertreter dieser Familie betrachtet. Nachdem die Expression und Aufreinigung von GlpG optimiert worden waren, wurden die NMR-Peaks der Methyl-Gruppen durch Punktmutationen zugeordnet. Diese wurden dazu genutzt lokale Änderungen der chemischen Umgebung und der Flexibilität der Seitenketten im Mikro- bis Millisekunden-Bereich für GlpG ohne und im Komplex mit Inhibitoren mittels Lösungs-NMR-Spektroskopie zu verfolgen. Die Ergebnisse deuten darauf hin, dass die Interaktion mit dem Inhibitor STS1775 Konformationsänderungen bzw. Änderungen in der Dynamik von Methyl-Gruppen induziert, die bis zu 18 Å weit vom aktiven Zentrum entfernt sind.Item Suppression of liquid-liquid phase separation and aggregation of antibodies by modest pressure application(2022-06-27) Fetahaj, Zamira; Jaworek, Michel W.; Oliva, Rosario; Winter, RolandThe high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid–liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity.Item Biophysikalische Einblicke in heterogene Modellbiomembranen, G-Quadruplexe und Eigenschaften der Flüssig-Flüssig-Phasentrennung von Peptiden(2023) Manisegaran, Magiliny; Winter, Roland; Kast, Stefan M.Die biophysikalische Chemie ist eine Forschungsdisziplin, die Antworten auf medizinische und biochemische Fragen liefert. Modellsysteme biologischer Makromoleküle werden verwendet, um ein umfassendes Verständnis biologischer Prozesse zu fördern. Dies kann Ansätze für die Diagnose und Behandlung von Krankheiten liefern. In dieser Arbeit werden physikalisch-chemische Einflüsse auf heterogene Modellbiomembranen, G-Quadruplexe und die Eigenschaften der Flüssig-Flüssig-Phasentrennung von Peptiden untersucht: Das erste Thema in der Arbeit ist der Einfluss des Immunregulators 25-Hydroxycholesterol auf das thermotrope und barotrope Phasenverhalten und die Struktur von Modellbiomembranen, das zweite die Bindungseigenschaften von Berberin an den G-Quadruplex der RG-1-Sequenz der RNA des SARS-CoV-2-Virus unter verschiedenen Lösungsbedingungen und das letzte Thema behandelt die Wechselwirkung zwischen zwei Phasenkoexistenzen verschiedener biologischer Systeme: Heterogene Membransysteme zeigen eine Phasenkoexistenz, die als Kompartimentierung der Zellmembran verstanden werden kann. Proteine und Nukleinsäuren können die Eigenschaft der Flüssig-Flüssig-Phasentrennung aufweisen.Item Qualitative and quantitative characterization of protein backbone heterogeneity by solid-state NMR spectroscopy(2023) Burakova, Ekaterina; Linser, Rasmus; Heise, HenrikeFlexibility of the polypeptide chains plays the key role in protein functions, their un-, re- and misfolding pathways. Understanding the conformational landscape which protein chain occupies statically and dynamically is essential for understanding of cellular processes and ultimately, designing efficient drugs, safe pesticides, and industrial biotechnological processes. NMR spectroscopy is an indispensable technique studying disordered molecules site-specifically, both in solution and in the solid state. Protein disorder covers the continuum between the static set of defined states and dynamic ensembles. Dynamic disorder can be converted into static disorder by freeze-trapping and studied in the solid phase. In solid-state NMR, static disorder manifests itself as the presence of additional peaks or, in the general case, severe line broadening. Converting the distribution of the resonance frequencies into conformational ensembles is not a trivial task due to the multitude of factors that contribute to the nuclear resonance frequencies. This works proposes approaches to analyze residue-specific static disorder by interpretation and quantification of heterogeneously broadened peaks in multidimentsional solid-state NMR spectra. The engineered routines reconstruct the distributions of the backbone dihedral angles φ and ψ on the basis of database analyses and by help of dihedral-angle predictors. The workflows are tested on a model sample as well as on a naturally heterogeneous functional amyloid (EAS∆15 rodlet sample), where the obtained heterogeneity scores are compared to those formed by peak shape parameters (widths, intensities, and tilt). The analysis of the EAS ∆15 sample demonstrates the intrinsic power and weaknesses of the proposed analysis for rather challenging systems where the only available high-resolution physico-chemical data are the peak shapes in the solid-state NMR spectra.Item Temperatur-, hydrostatische Druck- und Kosolvenseffekte auf Protein-Liganden-Systeme(2023) Jahmidi-Azizi, Nisrine; Winter, Roland; Czeslik, ClausProtein-Ligand-Interaktionen sind von grundlegender Bedeutung für sämtliche biochemische Prozesse und unverzichtbar für alle Lebensformen. Die molekulare Erkennung, die durch Spezifität und Affinität charakterisiert ist, spielt eine entscheidende Rolle in biologischen Prozessen wie der Selbstreplikation, dem Stoffwechsel und der Informationsverarbeitung. Die Anwendung von Druck auf Proteine beeinflusst ihre freie Energie und die Konformationslandschaft, was zur Entstehung verschiedener Konformationszustände mit unterschiedlicher Reaktivität führen kann. Studien, die den Einfluss von Druck auf biologische Systeme untersuchen, sind besonders relevant, da Organismen in den tiefen Ozeanen extrem hohem hydrostatischem Druck ausgesetzt sind. Da die Tiefsee als Quelle für vielfältige Lebensformen gilt, ist das Verständnis der Auswirkungen von Druck auf biologische Systeme von entscheidender Bedeutung, um das Leben und seine physikalischen Grenzen zu erforschen. Allerdings bestehen noch erhebliche Wissenslücken hinsichtlich der Auswirkungen von Stressfaktoren auf biomolekulare Prozesse wie Protein-Ligand-Interaktionen. In dieser Dissertation liegt der Fokus auf der Untersuchung der Auswirkungen von Druck, Temperatur und Kosolventen auf Protein-Ligand-Systeme. Dabei werden Erkenntnisse über die thermodynamischen Eigenschaften von Ligandenbindungsreaktionen gewonnen und Aspekte wie Packungseigenschaften, Strukturschwankungen, Interaktionsmechanismen und Veränderungen in der Hydration dieser Prozesse beschrieben.Item Characterization of protein structure and dynamics by solution and solid-state NMR(2023) Medina Gomez, Sara; Linser, Rasmus; Rauh, DanielThe study of proteins for understanding biological processes or as an approach to treating diseases has become more relevant in recent years. Different structural biology approaches can help to answer some of these questions, including nuclear magnetic resonance (NMR) spectroscopy, which provides information at atomic resolution about the structure and dynamics on a broad range of timescales. Traditionally, protein studies are carried out by solution NMR, where many methodologies have been developed over the years that allow one to obtain a great variety of information. However, in solution NMR studies, the protein size is limited to up to 50 kDa. Using different labeling schemes and different levels of protonation in the protein, with solid-state NMR and 1H detection, we can access biologically relevant systems with a molecular size larger than 50 kDa and extract information of their structure and dynamics on a different timescale with atomic resolution. This thesis presents a combination of solution and solid-state NMR methodologies to understand the dynamical processes of the MAP kinase p38α and their changes upon ligand binding as a tool for characterizing the allosteric dynamic network present in the protein. All this was done by combining different labeling schemes and proving new methodologies for acquiring relaxation dispersion experiments that can be used for large protein systems. Furthermore, relaxation dispersion techniques in solid-state NMR are used to characterize the modulation of dynamics by inter- and intramolecular interaction for the SH3 domain of chicken α-spectrin by introducing a mutation in the RT loop. Furthermore, this work presents the characterization of the secondary structural element for the N-terminal domain of RhoGDI1, a domain thought to be disordered in the apo form and ordered in complex with the GTPase client. However, with the experimental chemical-shift information, we proved it to have a secondary structure before complex formation.Item Struktur und Dynamik von Modellbiomembranen unter verschiedenen physikalischen und chemischen Bedingungen(2023) Kriegler, Simon; Winter, Roland; Czeslik, ClausPlasmamembranen umgeben Zellen und deren Organellen, weshalb sie in praktisch jedem Lebewesen vorkommen und wichtige biologische Funktionen übernehmen. Dabei fungieren sie als spezifische Barriere, die die Diffusion von Stoffen in und aus der Zelle regulieren. Biomembranen reagieren empfindlich auf Temperatur- und Druckänderungen sowie auf die Anwesenheit bestimmter Cosolventien. Diese Arbeit beschäftigt sich im Wesentlichen mit den Auswirkungen verschiedener Substanzen auf Modellbiomembranen in Abhängigkeit des Drucks und der Temperatur. In dieser Dissertation konnte gezeigt werden, dass in Bakterien vorkommende ungesättigte Lipide die auf dem Mars vorkommenden Umweltbedingungen überstehen, sodass prokaryotische Zellmembranen in den Salzlaken unterhalb des Südpols der Marsoberfläche überdauern könnten. Außerdem wurden die verschiedenen Anpassungen von Lipidmembranen an hohe hydrostatische sowie osmotische Drücke studiert. Die Kombination aus der Zugabe eines antimikrobiellen Peptids und einer Hochdruckbehandlung auf bakterielle Modellmembranen demonstriert einen neuen Ansatz zur Nahrungsmittelkonservierung. Ein weiteres Kapitel beschäftigt sich mit dem Einfluss bestimmter pseudo-Naturstoffe, die durch lysosomale Membranen diffundieren können, um die Aktivität der verdauenden Enzyme innerhalb von Lysosomen durch eine pH-Wert Änderung zu senken. Zuletzt befasst sich diese Arbeit mit dem Einbau von künstlichen Lipiden auf Imidazolbasis in verschiedene Phospholipidmembransysteme zur gezielten Manipulation derer Eigenschaften.Item Characterization of conformational heterogeneity via higher-dimensionality, proton-detected solid-state NMR(2022-09-23) Burakova, Ekaterina; Vasa, Suresh K.; Linser, RasmusSite-specific heterogeneity of solid protein samples can be exploited as valuable information to answer biological questions ranging from thermodynamic properties determining fibril formation to protein folding and conformational stability upon stress. In particular, for proteins of increasing molecular weight, however, site-resolved assessment without residue-specific labeling is challenging using established methodology, which tends to rely on carbon-detected 2D correlations. Here we develop purely chemical-shift-based approaches for assessment of relative conformational heterogeneity that allows identification of each residue via four chemical-shift dimensions. High dimensionality diminishes the probability of peak overlap in the presence of multiple, heterogeneously broadened resonances. Utilizing backbone dihedral-angle reconstruction from individual contributions to the peak shape either via suitably adapted prediction routines or direct association with a relational database, the methods may in future studies afford assessment of site-specific heterogeneity of proteins without site-specific labeling.Item Stabilität ausgewählter Flüssig-Flüssig- Phasentrennungen in biomolekularen Systemen(2023) Ostermeier, Lena; Winter, Roland; Czeslik, ClausDie Flüssig-Flüssig-Phasentrennung (englisch: liquid-liquid phase separation, LLPS) hat sich als Schlüsselmechanismus für die intrazelluläre Organisation erwiesen, wobei zahlreiche aktuellere Studien wesentliche Erkenntnisse über die Rolle der LLPS im Bereich der Zellbiologie geliefert haben. Es gibt zudem Hinweise darauf, dass die LLPS mit einer Reihe von Erkrankungen, einschließlich neurodegenerativer Krankheiten, in Verbindung gebracht werden kann. Die pathologische Aggregation des α-Synucleins, die in kausalem Zusammenhang mit der PARKINSON-Krankheit steht, kann durch Tröpfchenkondensation erfolgen, welche schließlich schrittweise in den amyloiden Zustand übergeht. Es wurde im Rahmen dieser Dissertation untersucht, wie der Fibrillierungsprozess des α-Synucleins, etwa durch die Zugabe des antimikrobiellen Peptids LL-III, beeinflusst werden kann. Darüber hinaus kann eine Flüssig-Flüssig-Phasentrennung auch zur Bildung von biomolekularen Kondensaten führen, welche als eine der anfänglichen Kompartimentierungsstrategien von Zellen gelten, die auch heute noch bei der Bildung von nicht-membranartigen Kompartimenten in biologischen Zellen vorherrschen. Daher wurden im Rahmen dieser Dissertation die Auswirkungen extremer Umweltbedingungen, insbesondere hochaggressiver Marssalze (Perchlorat und Sulfat) und hohen Drucks, auf die Bildung biomolekularer Proteinkondensate untersucht. Eine ähnliche Art der Tröpfchen, die wässrigen Zweiphasensysteme, können zudem mit biokatalytischen Reaktionen in Verbindung gebracht werden. Schließlich ist die gezielte Anpassung des Reaktionsmediums eine interessante Alternative zur Modulation der kinetischen Eigenschaften biokatalytischer Reaktionen. Deshalb wurde die kombinierte Wirkung eines wässrigen Zweiphasensystems und eines hohen hydrostatischen Drucks auf die Kinetik der FDH-katalysierten Oxidation von Formiat zu CO2 im Rahmen dieser Dissertation studiert.Item Flüssig-Flüssig-Phasenübergänge in biomolekularen Systemen(2022) Fetahaj, Zamira; Winter, Roland; Czeslik, ClausItem Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress(2022-06-24) Kamali, Armin; Jahmidi-Azizi, Nisrine; Oliva, Rosario; Winter, RolandBecause organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine-N-oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein.Item Tracing protein native radicals under in vitro and in vivo conditions via EPR(2022) Meichsner, Shari Lorraine; Kasanmascheff, Müge; Summerer, DanielAlthough more than a hundred thousand protein structures are registered in the Protein Data Bank, there are still more open questions than answers. One of these proteins that have captivated researchers for decades is ribonucleotide reductase, short RNR. Due to its central role in every living organism, RNR has been the focus of research on several occasions, but many questions remain unanswered despite numerous studies. Moreover, information about RNR structure in its natural environment, namely living cells, is entirely lacking. A method that can be used for protein structure elucidation is electron paramagnetic resonance, shortly EPR. EPR is also widely used for RNR studies since the enzyme has a native radical that is used for catalysis. In this work, state-of-the-art EPR spectroscopy was used to address some of the open questions related to RNR. Advanced EPR techniques were used to determine the structure of the stable tyrosyl radical Y122• in E. coli class Ia RNR in living whole E. coli cells at high resolution. Furthermore, EPR was used to investigate the in vivo radical distribution of RNR. In addition to in-cell EPR, three other applications of magnetic resonance spectroscopy are highlighted in this work. First, insights into the development process of a new spin label are given, using RNR as a model system. Through these studies, azidophenylalanine was identified as a suitable candidate that could contribute to the field of in-cell protein structure elucidation via spin labeling. Second, a high-pressure apparatus was used to study the effect of pressure on E. coli RNRs structure by EPR. Finally, independently of RNR, cw-EPR was used to characterize the long-lived C60 radical anion generated in a molecular coordination cage.Item Supplement to RISM-based pressure-dependant computational spectroscopy(2023) Pongratz, TimItem Rism-based pressure-dependent computational spectroscopy(2022) Pongratz, Tim; Kast, Stefan M.; Winter, RolandSpectroscopic measurements are an indispensable tool in chemical analysis; even under extreme conditions such as high hydrostatic pressures, they can provide valuable insights. Theoretical methods that can reliably reproduce observables in solution can be used to validate the obtained results. A common theoretical model is the Reference Interaction Site Model (RISM), which was used in this work. In the first part, a previously developed method for calculating IR frequencies with the embedded cluster(EC)-RISM under equilibrium conditions was extended to non-equilibrium thermodynamics for IR spectroscopy. The pressure-dependent IR frequency shifts of TMAO and the cyanide anion were investigated as model systems. Furthermore, EC-RISM was used here for the first time to calculate EPR observables at ambient conditions. First, experiments with the geometrically optimized structure showed that EC-RISM gives significantly better results than a standard continuum calculation despite a large deviation from the experiment. A significant improvement in the direction of the experimental values was achieved by using a large number of snapshots from an ab initio molecular dynamics simulation (AIMD) instead of a single geometry. In general, in the context of the theoretical description of high-pressure effects on proteins, the critical question can be raised whether using force fields parameterized for ambient conditions is appropriate for high-pressure conditions. To answer this question, the pressure dependence of the peptide backbone was investigated in the third part, and the small molecules N-methyl acetamide (NMA) and Ac-Gly/Ala-NHMe were used as model systems. In this work, it was shown that EC-RISM is a suitable method of choice for the calculation of spectroscopic observables in solution. Especially when non-ambient conditions are to be examined, EC-RISM shows its strength since it is relatively easily extensible, e.g., high-pressure environments.Item Temperatur-, hydrostatische Druck- und Kosolvenseffekte auf die Flüssig-Flüssig-Phasentrennung verschiedener Proteinsysteme(2022) Cinar, Hasan; Winter, Roland; Czeslik, ClausBereits vor 120 Jahren entstand die Idee, dass kondensierte, tropfenartige Granula zur Struktur des Protoplasmas beitragen. Jedoch wurde erst in den letzten 10 Jahren neben der durch "klassische" Lipid-Doppelschichtmembranen gestützte Kompartimentierung (z.B. Plasmamembran, Lysosomen, endoplasmatisches Retikulum, Mitochondrien) erkannt, dass die Flüssig-Flüssig-Phasentrennung (LLPS) von Proteinen und Nukleinsäuren eine wichtige Rolle bei der membranlosen Kompartimentierung von Zellen durch die Bildung von biomolekularen Kondensaten spielten. Solche Kondensationsprozesse sind mit zahlreichen intrazellulären Organismen an der subzellulären Organisation wie, P-bodies, Stressgranula, Keimgranula und Zentrosomen im Zytoplasma und Nukleoli, Chromatin und Cajal-Körper im Zellkern beteiligt. Unteranderem sind diese Kondensate an Membranfunktionen, wie beim Kernporenkomplex beteiligt oder man findet sie, wie im Fall der postsynaptischen Dichte, in der Nähe von Zellmembranen wieder. Im Bestreben, biomolekulare LLPS-Prozesse zu verstehen, konzentriert sich diese Arbeit auf die Gleichgewichtsthermodynamik der Flüssig-Flüssig-Phasentrennung unterschiedlicher Proteinsysteme in Abhängigkeit der Temperatur, des Druckes und unterschiedlicher Kosolvens-Bedingungen. Um die Funktion solcher LLPS-gesteuerten biologischen Kompartimentierungsvorgänge vollständig verstehen zu können, reicht jedoch die Kenntnis des Gleichgewichtsverhaltens solcher LLPS-Prozesse nicht aus. Aufgrund dessen wurde die Arbeit auf die Untersuchung der Phasenübergangskinetik der LLPS-Prozesse erweitert.