Physikalische Chemie

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Assignment of the N-terminal domain of mouse cGAS
(2025-01-04) Aucharova, Hanna; Linser, Rasmus
Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme that is a member of the nucleotidyltransferase (NTase) family and functions as a DNA sensor. The protein is comprised of a catalytic NTase core domain and an unstructured hypervariable N-terminal domain (NTD) that was reported to increase protein activity by providing an additional DNA-binding surface. We report nearly complete 1H, 15N, and 13C backbone chemical-shift assignments of mouse cGAS NTD (residues 5-146), obtained with a set of 3D and 4D solution NMR experiments. Analysis of the chemical-shift values confirms that the NTD is intrinsically disordered. These resonance assignments can provide the basis for further studies such as activation by DNA and protein-protein interactions.
Multiscale approximations of integral equation-based solvation models
(2025) Eisel, Lennart; Kast, Stefan M.; Czodrowski, Paul
Die Anwendbarkeit und Kosten quantenchemischer Solvatationsmodelle, wie dem "embedded cluster reference interaction site model" (EC-RISM) werden vor allem durch die Berechnungskosten der Elektronenstruktur, sowie des elektrostatischen Potentials bestimmt, bedingt durch die iterative Lösung dieser Verfahren und der damit einhergehenden Wiederholung der teuren Elektronenstrukturrechnung. Hierdurch wird ihre Anwendung auf chemische Systeme geringer Größe beschränkt. In dieser Arbeit wird ein neues Solvatationsmodell, ONIOM-EC-RISM, präsentiert mit dem dieser Rechenaufwand für das bisherige EC-RISM Verfahren, durch die Einführung einer multiskalen Approximation der Elektronenstruktur, drastisch reduziert werden kann. Hierdurch erschließt sich die Möglichkeit das EC-RISM-Solvatationsmodell auch auf größere chemische Systeme, wie z.B. Proteinsysteme, anzuwenden. Neben den für das Verständnis des ONIOM-EC-RISM-Modells relevanten theoretischen Grundlagen additiver und subtraktiver Multiskalenapproximationen, des statistisch-thermodynamischen RISM-Solvatationsmodells, sowie des methodisch verwandten ONIOM-PCM-Solvatationsmodells, werden die Theorie und technische Implementation des neuen Modells umfangreich dargestellt. Darüber hinaus wird aufgezeigt wie zuvor für das EC-RISM-Referenzmodell verwendete empirische Korrekturen in den ONIOM-EC-RISM-Kontext übertragen werden können. Hierbei wird das Größenextrapolationslimit der ONIOM-Methode ausgenutzt, wodurch Korrekturen erhalten werden, die frei von jeglichen Partitionierungsfehlern sind. Die resultierenden Modelle sind in der Lage, die $\pKa$-Vorhersagequalität des EC-RISM-Referenzmodells für den pKa-Datensatz der SAMPL6-Challenge, einem "blind prediction" Wettbewerb zur Vorhersage thermodynamischer Größen, zu reproduzieren und teilweise zu übertreffen, während gleichzeitig die Gesamtkosten des EC-RISM-Verfahrens drastisch reduziert werden können. Neben der Validierung des ONIOM-EC-RISM-Verfahrens anhand von pKa-Werten wird demonstriert, wie das ONIOM-EC-RISM-Modell zusätzlich zur Vorhersage chemischer Verschiebungen eines Pentapeptidsystem verwendet werden kann. In diesem Zusammenhang wird ein neuartiger Ansatz vorgestellt der es erlaubt, pH-abhängige chemische Verschiebungen direkt aus spektroskopischen sowie pKa-Vorhersagen zu berechnen. Dies eröffnet erstmalig die Möglichkeit der direkten Modellierung von NMR-Titrationsexperimenten auf Grundlage des EC-RISM-Solvatationsmodells.
Evolved readers of 5-carboxylcytosine CpG dyads reveal a high versatility of the methyl-CpG-binding domain for recognition of noncanonical epigenetic marks
(2024-01-29) Kosel, Brinja; Bigler, Katrin; Buchmuller, Benjamin C.; Acharyya, Suchandra R.; Linser, Rasmus; Summerer, Daniel
Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5-methylation (mC), a total of four different 5-modifications can theoretically co-exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5-carboxylcytosine (caC) dyads based on the methyl-CpG-binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC-dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design.
Nonlinear impact of electrolyte solutions on protein dynamics
(2024-02-23) Daronkola, Hosein Geraili; Söldner, Benedikt; Singh, Himanshu; Linser, Rasmus; Vila Verde, Ana
Halophilic organisms have adapted to multi-molar salt concentrations, their cytoplasmic proteins functioning despite stronger attraction between hydrophobic groups. These proteins, of interest in biotechnology because of decreasing fresh-water resources, have excess acidic amino acids. It has been suggested that conformational fluctuations – critical for protein function – decrease in the presence of a stronger hydrophobic effect, and that an acidic proteome would counteract this decrease. However, our understanding of the salt- and acidic amino acid dependency of enzymatic activity is limited. Here, using solution NMR relaxation and molecular dynamics simulations for in total 14 proteins, we show that salt concentration has a limited and moreover non-monotonic impact on protein dynamics. The results speak against the conformational-fluctuations model, instead indicating that maintaining protein dynamics to ensure protein function is not an evolutionary driving force behind the acidic proteome of halophilic proteins.
Transient structural properties of the Rho GDP‐dissociation inhibitor
(2024-06-09) Medina Gomez, Sara; Visco, Ilaria; Merino, Felipe; Bieling, Peter; Linser, Rasmus
Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases’ mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.
Allostery at a protein-protein interface harboring an intermolecular motional network
(2024-08-19) Medina Gomez, Sara; Gonzalez, Tye I.; Vasa, Suresh K.; Linser, Rasmus
Motional properties of proteins govern recognition, catalysis, and regulation. The dynamics of tightly interacting residues can form intramolecular dynamic networks, dependencies fine-tuned by evolution to optimize a plethora of functional aspects. The constructive interaction of residues from different proteins to assemble intermolecular dynamic networks is a similarly likely case but has escaped thorough experimental assessment due to interfering association/dissociation dynamics. Here, we use fast-MAS solid-state 15N R1ρ NMR relaxation dispersion aided by molecular-dynamics simulations to mechanistically assess the hierarchy of individual μs timescale motions arising from a crystal-crystal contact, in the absence of translational motion. In contrast to the monomer, where particular mutations entail isolated perturbations, specific intermolecular interactions couple the motional properties between distant residues in the same protein. The mechanistic insights obtained from this conceptual work may improve our understanding on how intramolecular allostery can be tuned by intermolecular interactions via assembly of dynamic networks from previously isolated elements.
Activation of a secondary-messenger receptor via allosteric modulation of a dynamic conformational ensemble
(2025-08-06) Söldner, Benedikt; Singh, Himanshu; Akoury, Elias; Witte, Gregor; Linser, Rasmus
Bacterial signaling cascades have recently become of great relevance in the context of bacterial antibiotics resistance. Cyclic diadenylate monophosphate (c-di-AMP) is a key bacterial secondary messenger involved in growth, biofilm formation, virulence gene expression and others. The activation mechanisms of c-di-AMP receptors like the trimeric PII-like proteins upon messenger binding have, however, remained elusive due the pivotal role of highly flexible protein regions. Here, using solution NMR spectroscopy to elucidate the interplay between the ordered and disordered structural elements of the apo and messenger-bound forms of the 44 kDa homotrimeric PII-like signal transduction protein A (PstA), we reveal a sensitive modulation of the conformational ensemble of those extended loops thought to bind the downstream interaction partners by messenger association at the receptor core. The orchestration of the spatial properties of the loops, despite their retained internal dynamics, reveals the importance of allosteric effects even for disordered structural elements, whose steerable ensemble properties have long escaped the classical structural-biology understanding.
Characterisation of protein structure and dynamics by NMR spectroscopy and computational methods
(2025) Söldner, Benedikt; Linser, Rasmus; Schäfer, Lars
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying the structure and dynamics of proteins. In contrast to almost all other experimental techniques, NMR spectroscopy facilitates the elucidation of site-specific protein dynamics on various timescales, making it an indispensable tool for structural biology of proteins. In the first chapter, the theory of NMR spectroscopy is introduced and an overview of frequently used NMR spectroscopic methods for studying protein structure and dynamics is given. In addition, an introduction about molecular dynamics (MD) simulations, a technique for studying protein dynamics on an atomic scale used for explaining the dynamics detected by NMR spectroscopy as well as constituting a technique for structure determination of proteins based on observables from NMR spectroscopy, is given. In chapter 2 – 5, the four major projects investigated for my Ph. D. are presented. In chapter 2, a newly developed method for determining accurate distances from 1H-detected solid-state NMR spectroscopy is presented and demonstrated by structure determination and restrained MD simulations of the chicken α-spectrin SH3 domain. In the project presented in chapter 3, microsecond-timescale dynamics of a small-molecule ligand bound to the active site of the human carbonic anhydrase II (hCAII) was revealed with solid-state NMR spectroscopy, where my contribution consisted in determining the origin of the dynamics on an atomic level using MD simulations. In the project shown in chapter 4, the influence of salt concentration on the protein dynamics was investigated by NMR spectroscopy and MD simulations. In the project presented in chapter 5, the secondary-messenger-induced allosteric modulation of conformational loop dynamics in the PII-like protein A (PstA) is investigated. In addition to the modulation of the spatial properties of the 30-residue long loops, in absence of the ligand, also slow µs-ms timescale dynamics in the core of PstA are revealed.
Strukturuntersuchungen an Proteinen und Protein-Lipid-Systemen mittels Infrarot-Spektroskopie und Röntgenbeugung
(2006-02-27T13:26:05Z) Kraineva, Julia; Winter, Roland; Geiger, Alfons
Zellfunktionen 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.
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, Rasmus
With 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.
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.
Scaffold-diverse synthesis via Petasis–sequence reactions and the discovery of IRE1α modulators
(2025) Avathan Veettil, Amrutha Krishnan; Waldmann, Herbert; Wu, Peng
Small 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.
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.
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, Thorben
Attaching 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.
5D solid-state NMR spectroscopy for facilitated resonance assignment
(2023-11-09) Klein, Alexander; Vasa, Suresh K.; Linser, Rasmus
1H-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.
Enhancing EPR capabilities: From 19F-ENDOR refinement to extreme condition measurements and sensitivity improvements
(2025) Schumann, Simon Lennard; Kasanmascheff, Müge; Clever, Guido
Electron 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.
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, Rasmus
The 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.
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, Müge
DNA 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.
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, Roland
The 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.
Bacterial model membranes under the harsh subsurface conditions of Mars
(2023-10-17) Tortorella, Attila; Oliva, Rosario; Giancola, Concetta; Petraccone, Luigi; Winter, Roland
Biomembranes 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.

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