Physikalische Chemie
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Item Strukturuntersuchungen an Proteinen und Protein-Lipid-Systemen mittels Infrarot-Spektroskopie und Röntgenbeugung(2006-02-27T13:26:05Z) Kraineva, Julia; Winter, Roland; Geiger, AlfonsZellfunktionen werden durch molekulare Elementarschritte gesteuert, die auf die Wechselwirkungen von Proteinen mit Proteinen, Lipiden, Nukleinsäuren und Kohlenhydraten beruhen. Das zelluläre Netzwerk mit seiner enormen funktionellen Vielfalt wird durch eine Vielzahl von schwachen und reversiblen Wechselwirkungen zwischen den einzelnen Reaktionspartnern ermöglicht. Diese Forschungsarbeit soll zum verbesserten Verständnis solcher Interaktionen beitragen. Der erste Themenbereich befasst sich mit der Aufklärung des Aggregationsmechanismus von amyloidogenen Proteinen am Beispiel von Prion-Protein, Transthyretin und Insulin. Im Fall der Amyloidosen liegt der pathologische Effekt darin, dass eine fehlerhaft gefaltete Isoform des Proteins gebildet wird, die in Form von Aggregaten in den Zellen abgelagert wird. Prion- und Transthyretin-Ablagerungen führen dann zu neurodegenerativen Erkrankungen des ZNS, bei Insulin bilden sich Ablagerungen in den Muskeln und führen dann zu deren Degeneration. Die Aufklärung des Auslösemechanismus der Amyloidosis, der Struktur und der Stabilität der fehlgefalteten Protein-Aggregate sind nicht nur vom großen akademischen Interesse, sondern auch entscheidend für die Entwicklung von therapeutischen Maßnahmen gegen diese, bis jetzt unheilbare, Erkrankungen. Molekulare Aggregationsmechanismen werden hier in vitro bei unterschiedlichen physikalischen Bedingungen wie Temperatur, Druck und pH-Wert mittels FTIR-Spektroskopie untersucht. Besonderes Interesse gilt dabei der Aufklärung der Sekundärstruktur der Intermediate und der Protein-Aggregate. Die Beteiligung von anderen molekularen Komponenten, wie DNA, an der Amyloidose wird ebenfalls berücksichtigt und analysiert. Lipid-Polymorphismus ist das Thema des zweiten Projekts. Schichten und Grenzflächen sind allgegenwärtig in biologischen Systemen. Hierzu zählen Zellmembranen, die viele strukturelle Aufgaben besitzen und eine Vielzahl an biologischen Funktionen ausführen. Besonderes Interesse gilt hier der Membran-Fusion, die ein äußerst wichtiger Prozess aller Zellen ist. Wässrige Monoolein-Dispersionen stellen einfache Modellsysteme dar, in denen Übergänge zwischen lamellaren und kubischen Phasen in vitro beobachtet werden können. Es besteht nämlich ein enger Zusammenhang zwischen der Struktur einer Fusions-Pore und inversen kubischen Phasen. Der Mechanismus und die Kinetik der Phasen-Umwandlungen werden mittels zeitaufgelöster Röntgenbeugung studiert. Ein dritter Themenbereich befasst sich mit der Wechselwirkung von Proteinen mit nanostrukturierten Lipidsystemen. Dabei steht der Einbau von Proteinen (Cytochrom c, α-Chymotrypsin und Insulin) in die kubischen Phasen von Monoolein im Mittelpunkt des Interesses. Kubische Phasen sind „weiche“ Analoga von Zeolithen, die hydrophile, hydrophobe und amphiphile Substanzen lösen können und als Transportmittel für Wirkstoffe oder als Matrix für die Protein-Kristallisation verwendet werden können. Der Einfluss der Proteine auf die strukturellen Eigenschaften der Lipid-Umgebung sowie die Stabilität der in die engen Geometrien eingeschlossenen Proteine werden mittels FTIR-Spektroskopie und Röntgenbeugung untersucht.Item Protein deuteration via algal amino acids to circumvent proton back-exchange for 1H-detected solid-state NMR(2024-02-21) Aucharova, Hanna; Klein, Alexander; Medina Gomez, Sara; Söldner, Benedikt; Vasa, Suresh K.; Linser, RasmusWith perdeuteration, solid-state NMR spectroscopy of large proteins suffers from incomplete amide-proton back-exchange. Using a 72 kDa micro-crystalline protein, we show that deuteration exclusively via deuterated amino acids, well-established in solution to suppress sidechain protonation without proton back-exchange obstacles, provides spectral resolution comparable to perdeuterated preparations at intermediate spinning frequencies.Item The accuracy limit of chemical shift predictions for species in aqueous solution(2024-02-05) Maste, Stefan; Sharma, Bikramjit; Pongratz, Tim; Grabe, Bastian; Hiller, Wolf; Erlach, Markus Beck; Kremer, Werner; Kalbitzer, Hans Robert; Marx, Dominik; Kast, Stefan M.Interpreting NMR experiments benefits from first-principles predictions of chemical shifts. Reaching the accuracy limit of theory is relevant for unambiguous structural analysis and dissecting theoretical approximations. Since accurate chemical shift measurements are based on using internal reference compounds such as trimethylsilylpropanesulfonate (DSS), a detailed comparison of experimental with theoretical data requires simultaneous consideration of both target and reference species ensembles in the same solvent environment. Here we show that ab initio molecular dynamics simulations to generate liquid-state ensembles of target and reference compounds, including explicitly their short-range solvation environments and combined with quantum-mechanical solvation models, allows for predicting highly accurate 1H (∼0.1–0.5 ppm) and aliphatic 13C (∼1.5 ppm) chemical shifts for aqueous solutions of the model compounds trimethylamine N-oxide (TMAO) and N-methylacetamide (NMA), referenced to DSS without any system-specific adjustments. This encompasses the two peptide bond conformations of NMA identified by NMR. The results are used to derive a general-purpose guideline set for predictive NMR chemical shift calculations of NMA in the liquid state and to identify artifacts of force field models. Accurate predictions are only obtained if a sufficient number of explicit water molecules is included in the quantum-mechanical calculations, disproving a purely electrostatic model of the solvent effect on chemical shifts.Item Scaffold-diverse synthesis via Petasis–sequence reactions and the discovery of IRE1α modulators(2025) Avathan Veettil, Amrutha Krishnan; Waldmann, Herbert; Wu, PengSmall molecules have long been central to pharmaceutical research, serving as therapeutic agents and effective tools for investigating biological functions. While structure-based and high-throughput screening approaches aid in drug discovery, the key challenge remains the limited exploration of chemical space, as most available small molecule libraries are dominated by flat, sp2-rich compounds. The growing number of potential therapeutic targets, driven by advances in genomics and proteomics, underscores the urgent need for novel chemistry strategies to generate diverse, structurally complex small molecules. The initial part of this thesis presents the synthesis of a diverse array of small molecules featuring polycyclic scaffolds with a high degree of sp3-hybridized carbon atoms and multiple stereogenic centers. This was achieved through three-component Petasis reaction (3C-PR) followed by intramolecular Diels–Alder reactions, as well as a sequential 3C-PR, ruthenium-catalyzed ring-closing metathesis (RCM), and intermolecular Diels–Alder reaction. Our synthetic efforts led to the formation of a collection of epoxyisoindoles and pyridazino[4,3-c]azepines in good yields, within 2-3 steps. The stereochemistry of the products was confirmed via single-crystal X-ray diffraction analysis. Our findings highlight the broad substrate scope and versatility of Petasis–sequence reactions in accessing previously unexplored polycyclic scaffolds with favorable predicted drug-like properties and potential biological relevance. The subsequent chapter focuses on the dual kinase and endoribonuclease (RNase) enzyme IRE1α, a key regulator of the unfolded protein response (UPR) and RNA metabolism. Dysregulation of the IRE1α–XBP1 axis of UPR has been implicated in the pathogenesis of multiple diseases, making IRE1α an attractive therapeutic target. A screening of a structurally diverse in-house compound library is conducted to identify new small molecule modulators of IRE1α RNase. This led to the discovery of two distinct classes of modulators: indole-based allosteric inhibitors that bind to the ATP binding pocket to suppress RNase activity, and aminopyrimidine-based activators that enhance RNase function while inhibiting its kinase activity. These compounds were further structurally modified and optimized to generate analogues with improved potency. Notably, inhibitor 54 exhibited IC50 values of 16 nM (IRE1α) and 9 nM (p-IRE1α) while activator 91 demonstrated EC50 values of 480 nM (IRE1α) and 180 nM (p-IRE1α). Comprehensive biophysical assays, mechanistic studies, and cellular evaluations support the therapeutic potential of these small molecules as modulators of IRE1α activity. Together, this work demonstrates the capability of Petasis–sequence reactions as efficient and complexity-generating strategies in constructing polycyclic bioactive small molecules and presents new molecular tools for exploring RNA-targeted therapeutic strategies, particularly through the modulation of IRE1α RNase activity.Item Triplet vinylidenes based on (benz)imidazole and 1,2,3-triazole N-heterocycles(2025-06-05) Kutin, Yury; Reitz, Justus; Drosou, Maria; Antoni, Patrick W.; He, Yijie; Selve, Victor R.; Boschmann, Sergius; Savitsky, Anton; Pantazis, Dimitrios A.; Kasanmascheff, Müge; Hansmann, Max M.Triplet vinylidenes, a new class of carbon-centered diradicals containing a monosubstituted carbon atom, remain largely unexplored. A series of triplet vinylidenes based on five-membered heterocycles, featuring 2- and 4-imidazole, benzimidazole as well as 1,2,3-triazole backbones, are generated upon irradiation of stable diazoalkenes and are investigated by electron paramagnetic resonance (EPR) spectroscopy. While the calculated S/T gaps strongly vary (∼9.9–18.4 kcal/mol), the experimental zero-field splitting (ZFS) D values are positioned in a rather narrow and characteristic range of D ∼ 0.366–0.399 cm–1. Electron nuclear double resonance (ENDOR) studies with 13C-labeled samples combined with quantum chemical calculations reveal a common motif of Aiso(13C) ≈ 50 MHz for the electronic structure of the vinylidene class. EPR decay experiments confirm that steric and electronic tuning of the heterocycle can hinder C–H activation pathways leading to the highest reported stabilities of up to 150 K. Quantum chemical studies elucidate and contrast plausible C–H insertion pathways, identifying an early triplet-to-singlet spin surface transition as the key factor that governs the stability of the vinylidenes.Item Labelizer: systematic selection of protein residues for covalent fluorophore labeling(2025-05-04) Gebhardt, Christian; Bawidamann, Pascal; Spring, Anna-Katharina; Schenk, Robin; Schütze, Konstantin; Moya Muñoz, Gabriel G.; Wendler, Nicolas D.; Griffith, Douglas A.; Lipfert, Jan; Cordes, ThorbenAttaching fluorescent dyes to biomolecules is essential for assays in biology, biochemistry, biophysics, biomedicine and imaging. A systematic approach for the selection of suitable labeling sites in macromolecules, particularly proteins, is missing. We present a quantitative strategy to identify such protein residues using a naïve Bayes classifier. Analysis of >100 proteins with ~400 successfully labeled residues allows to identify four parameters, which can rank residues via a single metric (the label score). The approach is tested and benchmarked by inspection of literature data and experiments on the expression level, degree of labelling, and success in FRET assays of different bacterial substrate binding proteins. With the paper, we provide a python package and webserver (https://labelizer.bio.lmu.de/), that performs an analysis of a pdb-structure (or model), label score calculation, and FRET assay scoring. The approach can facilitate to build up a central open-access database to continuously refine the label-site selection in proteins.Item 5D solid-state NMR spectroscopy for facilitated resonance assignment(2023-11-09) Klein, Alexander; Vasa, Suresh K.; Linser, Rasmus1H-detected solid-state NMR spectroscopy has been becoming increasingly popular for the characterization of protein structure, dynamics, and function. Recently, we showed that higher-dimensionality solid-state NMR spectroscopy can aid resonance assignments in large micro-crystalline protein targets to combat ambiguity (Klein et al., Proc. Natl. Acad. Sci. U.S.A. 2022). However, assignments represent both, a time-limiting factor and one of the major practical disadvantages within solid-state NMR studies compared to other structural-biology techniques from a very general perspective. Here, we show that 5D solid-state NMR spectroscopy is not only justified for high-molecular-weight targets but will also be a realistic and practicable method to streamline resonance assignment in small to medium-sized protein targets, which such methodology might not have been expected to be of advantage for. Using a combination of non-uniform sampling and the signal separating algorithm for spectral reconstruction on a deuterated and proton back-exchanged micro-crystalline protein at fast magic-angle spinning, direct amide-to-amide correlations in five dimensions are obtained with competitive sensitivity compatible with common hardware and measurement time commitments. The self-sufficient backbone walks enable efficient assignment with very high confidence and can be combined with higher-dimensionality sidechain-to-backbone correlations from protonated preparations into minimal sets of experiments to be acquired for simultaneous backbone and sidechain assignment. The strategies present themselves as potent alternatives for efficient assignment compared to the traditional assignment approaches in 3D, avoiding user misassignments derived from ambiguity or loss of overview and facilitating automation. This will ease future access to NMR-based characterization for the typical solid-state NMR targets at fast MAS.Item Enhancing EPR capabilities: From 19F-ENDOR refinement to extreme condition measurements and sensitivity improvements(2025) Schumann, Simon Lennard; Kasanmascheff, Müge; Clever, GuidoElectron paramagnetic resonance (EPR) spectroscopy is a technique with many different application fields. It is gaining popularity in medicine, material science, and biochemistry. As EPR was further established in other research fields, several new methodologies arose. Over the years, methods have been developed to detect interactions between two paramagnetic centers and a paramagnetic center and a magnetic nucleus. These diverse methodologies allow for structural and function analysis through distance measurements and coupling analysis. The need for higher precision measurements of minimal distances grew, and methods were developed and employed to satisfy this need. This thesis modifies 19F ENDOR measurements for very short distances from 94 GHz to 34 GHz, enhancing the technique's accessibility for a broader scientific audience. It also investigates DNA G-quadruplexes (GQ), which are critical to essential biological processes such as telomerase maintenance and gene expression. This research showcases the successful application of the 19F-ENDOR methodology at 34 GHz, overcoming the limitations posed by the complexity and scarcity of higher-frequency spectrometers. Notably, the approach retains sensitivity and orientational resolution, enhancing our understanding of GQs and expanding the methodological toolbox for studying other macromolecules. Furthermore, analyzing biological processes sometimes means looking outside the established boundaries. In some cases, life exists in extreme environments that are not easily reproduced in lab scenarios, like high-pressure deep-sea environments, and are not always reliant on abundant amounts of substances; in some cases, a low amount of molecules can already change biological function. Both of these edge cases are not easily accessible for EPR spectroscopy. A robust high-pressure EPR setup for pressures up to 4 kbar was constructed and tested during this thesis. This not only allows for basic EPR experiments but also opens the door to the full variety of dipolar spectroscopy methods available in EPR by following an out-of-spectrometer approach. This allows the application to be independent of the later spectrometer setup, simplifying the application drastically. Additionally, a high-sensitivity resonator with an extra large sample entrance for microwave and radio frequency double resonance experiments was built and established to allow for measurements of very low-concentration samples that were not feasible in a timely manner with commercially available resonators.Item Microsecond timescale conformational dynamics of a small‐molecule ligand within the active site of a protein(2023-11-16) Kotschy, Julia; Söldner, Benedikt; Singh, Himanshu; Vasa, Suresh K.; Linser, RasmusThe possible internal dynamics of non-isotope-labeled small-molecule ligands inside a target protein is inherently difficult to capture. Whereas high crystallographic temperature factors can denote either static disorder or motion, even moieties with very low B-factors can be subject to vivid motion between symmetry-related sites. Here we report the experimental identification of internal μs timescale dynamics of a high-affinity, natural-abundance ligand tightly bound to the enzyme human carbonic anhydrase II (hCAII) even within a crystalline lattice. The rotamer jumps of the ligand's benzene group manifest themselves both, in solution and fast magic-angle spinning solid-state NMR 1H R1ρ relaxation dispersion, for which we obtain further mechanistic insights from molecular-dynamics (MD) simulations. The experimental confirmation of rotameric jumps in bound ligands within proteins in solution or the crystalline state may improve understanding of host-guest interactions in biology and supra-molecular chemistry and may facilitate medicinal chemistry for future drug campaigns.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.