Universität Stuttgart
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Item Open Access Mechanistic studies on the DNA methyltransferases DNMT3A and DNMT3B(2021) Dukatz, Michael; Jeltsch, Albert (Prof. Dr.)In this work, both regulatory and catalytic mechanisms of de novo methyltransferases were investigated, which include interactions with other proteins and the specific recognition of the substrate sequence. Another part of this work strived to elucidate how enzymatic generation of 3-methylcytosine by DNMT3A can occur.Item Open Access Mechanistic study on the DNA methyltransferase DNMT3A(2024) Kunert, Stefan; Jeltsch, Albert (Prof. Dr.)Item Open Access Squalene-Hopene cyclase catalyzed isomerization of monoterpenes(2020) Diether, Svenja; Hauer, Bernhard (Prof. Dr.)Item Open Access Gold nanoparticle-mediated DNA origami nanoarchitectures(2024) Peil, Andreas; Na Liu, Laura (Prof. Dr.)Since its origin in the 1980s, DNA (deoxyribonucleic acid) nanotechnology has established itself as a captivating nanofabrication technique with ever increasing impact that combines aspects from physics, chemistry, and biology to construct artificial nanosystems by means of molecular self assembly. Within the field of DNA nanotechnology, the DNA origami technique represents one of the most versatile fabrication tools to craft functional two-dimensional (2D) and three-dimensional (3D) nanostructures from the bottom up. These structures offer precisely tailored geometries along with programmable functions, featuring positional addressability with sub-5 nm resolution and exceptional spatiotemporal accuracy. This thesis discusses strategies to employ the DNA origami technique to assemble intricate hybrid nanosystems with synergistically integrated gold nanoparticles (AuNPs). The AuNPs take over different roles; they grant (i) structural and (ii) functional features and enable the (iii) optical monitoring of the systems. This approach allows the fabrication of nanostructures piece by piece to explore their structural and functional properties at the nanoscale in detail. The first publication covers different strategies for the hierarchical assembly of topological DNA origami structures using a AuNP-templated self-assembly approach. The assembly of [2], [3], and [4]catenanes with interconnecting AuNPs is elucidated. The AuNPs can be controllably released to disconnect the individual rings, leaving only the mechanical bond of the catenane chain. In the second publication, a dynamic AuNP-DNA origami gear system is presented that is designed to emulate a planetary gearset with precise spatiotemporal control over its rotation dynamics. The AuNPs serve three crucial tasks. They (i) structurally link the origami ring modules, (ii) mediate the rotation and (iii) enable the real time optical tracking of the rotation via fluorescence spectroscopy. The system enables tightly orchestrated and programmable bidirectional rotations. In the third publication, reconfigurable chiral metastructures comprising multiple plasmonic particles that are accurately positioned in a helical manner around a DNA origami template are discussed. The implementation of a DNA ‘swingarm strategy’ enables the simultaneous and efficient relocation of multiple closely spaced AuNPs over large distances to precisely tune the chiroptical response of the system. The presented publications illustrate the beneficial synergies between DNA origami systems and rationally integrated AuNPs with the aim to advance and expand the application spectrum of these hybrid nanosystems within their scientific disciplines.Item Open Access Physikalische und chemische Wechselwirkungen in Gelatine-Methacryloyl-Lösungen und deren Vernetzung zu Hydrogelen als Trägerstruktur für Gelenkknorpel-Äquivalente(2021) Rebers, Lisa; Tovar, Günter (Prof. Dr.)Item Open Access Peptide und Fusionsproteine für die Biomineralisation von Hydroxylapatit(2021) Henkes, Thorsten Matthias; Hauer, Bernhard (Prof. Dr.)Mittels des sogenannten Phagen Display wurden Peptide identifiziert, welche an Hydroxylapatit binden. Diese Bindemotive wurden in oberflächenaktive Fusionsproteine integriert. Die Bindung der Phagen-präsentierten Peptide, von synthetischen Peptiden und der Fusionsproteine an Hydroxylapatit sowie der Einfluss von Peptiden und Fusionsproteinen auf die Nukleation von Hydroxylapatit wurden untersucht. Ebenso wurden gebildete Präzipitate mittels SEM EDX und TEM charakterisiert. Auf diese Weise wurden Peptidmotive und Fusionsproteine identifiziert, welche die Nukleation von Hydroxylapatit beschleunigen oder verlangsamen können.Item Open Access Biochemical characterization of protein lysine methyltransferases-regulation, specificity and effect of somatic cancer mutants(2023) Khella, Mina S.; Jeltsch, Albert (Prof. Dr.)Item Open Access Enzymkatalysierte regioselektive N-Methylierung und N-Alkylierung von Pyrazolen(2021) Bengel, Ludwig L.; Hauer, Bernhard (Prof. Dr.)Item Open Access Biochemical investigations of multivalent chromatin reading domains(2024) Choudalakis, Michel; Jeltsch, Albert (Prof. Dr.)In eukaryotes, the negatively charged nuclear DNA wraps around cationic histone proteins to form nucleosomes and compact the genetic information. Histones carry several post-translational modifications (PTMs) that appear in combinatorial patterns. These marks are interpreted by non-covalent interactions with proteins containing histone modification interacting domains (HiMIDs), also known as “reader” domains. Thirty years ago, it was proposed that the histone marks act as signals in the regulation of transcription and other chromatin functions. With time, this concept has been refined to suggest that combinatorial patterns of marks represent context-specific signals, termed a 'histone code'. It functions as one of the epigenetic regulatory mechanisms, which control reversible and heritable changes in cellular phenotype. Intermolecular models demonstrate thermodynamic benefits from multivalent engagement of nucleosomes, suggesting their widespread occurrence. However, so far only few multivalent readers are known and dissecting their function has been very challenging. This thesis focuses on HiMIDs with complex roles that simultaneously interact with two histone PTMs or two different substrates. Introducing the theoretical foundation, I discuss the thermodynamic and biological basis of how multivalent interactions can guide effector protein complexes, targeting their functions to distinct regions and chromatin states. Then, I present data from the characterisation of the readers DNMT3A-PWWP, DDX19A, and UHRF1-TTD in the context of multivalent engagement of histone PTMs and biomolecules. Starting with DNMT3A-PWWP, I quantified the binding of the wild-type (WT) and a mutant domain to histone H3K36me2/3 peptides, showing negligible differences, while my colleagues showed that the mutant has drastically reduced binding to DNA and nucleosomal substrates. I, then, studied the R-loop helicase DDX19A to demonstrate a very strong binding to H3K27me3 peptides in the nanomolar range, complementing the findings of a complex functional study. The latter showed that interaction with H3K27me3 is necessary for robust DDX19A-mediated R-loop resolution, and LSD1-target gene silencing. With UHRF1-TTD, I discovered and quantified its preferential binding to H3K4me1-K9me2/3 peptides vs H3K9me2/3 alone and engineered mutants with specific and differential binding changes leading to the discovery of a novel Kme1 read-out mechanism, based on the interaction of R207 methylene groups with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. High-throughput sequencing (HTS) data revealed strong TTD binding at chromatin sites with H3K4me1 peaks and broad H3K9me2/3 signal, which are enriched on enhancers and promoters of cell-type specific genes at the flanks of cell-type specific transcription factor binding sites. Data from the full-length protein in mouse and human cells evidenced the physiological role of the H3K4me1-K9me2/3 double marks in TTD-mediated UHRF1 recruitment. To further illustrate this point, I investigated UHRF1-dependent silencing of repeat elements (RE). To this end, I developed RepEnTools, improving the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, with carefully chosen and validated settings, enhancing accessibility, and adding some key functions. RepEnTools analyses showed that chromatin binding of hUHRF1-TTD and full-length mUHRF1 was strongly enriched on different REs promoters with the H3K4me1-K9me3 double mark where UHRF1 represses their expression. The data suggest a novel functional role for the H3K4me1-K9me3 signal of the histone code that is both sequence independent and conserved in two distinct mammals. Taken together, the work presented here is consistent with and supports the histone code theory, best illustrated by UHRF1-TTD which binds a specific double mark that has a biological meaning going beyond the meaning of the individual marks. In this thesis, I presented various mechanisms that influence epigenomic regulation, including chromatin 3D-architecture, accessibility, transcription factor recruitment, and chromatin marks. Especially in the context of UHRF1-TTD functions, I discussed how DNA, RNA, histones, and covalent modifications thereof interweave to produce the signalling network necessary throughout the lifetime of the mammalian cell, during differentiation, development and every other phase of life. Thus, within the three-dimensional scaffold of chromatin structures these biomolecules and their modifications collectively form the context-specific network of effectors and maintainers of the epigenomic modifications. The ways in which they influence transcription and translation are only now becoming unravelled. Hence, the recent data suggest the existence of not just a histone code, but a 3D-chromatin modification code, which dictates how biomolecules and their modifications collectively implement epigenomic regulation by interactions along the chromatin and through 3D space. As shown in these projects, readers commonly use the mechanism of multivalent interactions to interpret such contextual signals and guide epigenomic effectors to their targets. The tools and workflows that were developed and applied in this work can be employed to reveal more instances of refined read-out among HiMIDs. Additionally, I leveraged my experience with fluorescence spectroscopy and made contributions to another two published studies. The first study demonstrated that the DNMT3A-ADD Zn-finger domain, which is a known H3K4me0 reader, also binds to a domain from the MECP2 protein. The association was quantified, and the specificity demonstrated with a binding deficient triple mutant. This interaction offers complex additional regulation options to DNMT3A and MECP2, in interplay with the histone code. The second study focused on SETD2, a H3K36me3 depositing enzyme, and the mechanism of its preference for a designed “super substrate” peptide. By elegantly combining computational simulations and experimental data, the study demonstrated that an H3 peptide substrate predominantly exists in an extended conformation in solution, while the super substrate forms a hairpin conformation. Upon binding to the enzyme, the hairpin is opened and the super substrate adopts a similar conformation as the canonical substrate. These results highlighted the dynamic nature of solubilised peptides' conformations, their impact on protein-protein interactions, and the significance of dynamic conformational changes in interactions.Item Open Access Scale-up of gas fermentations : modelling tools for risk minimisation(2020) Siebler, FloraThe reduction of greenhouse gas emissions is a global endeavour supported by society, politics and industry. In recent years, circular economy, reducing the exploitation of fossil energy sources, have increased the demand for new solutions when producing commodities and fine chemicals. Caboxydotrophic fermentations with acetogenic bacteria are potential processes in order to reach these goals. They convert gaseous substrates such as CO, and CO2/H2 mixtures. However, gases as sole substrate are rather challenging, not only in small lab-scales but especially in large-scale. Transferring an efficient fermentation process from experimental to industrial scales often results in unpredictable performance losses. This study presents an in silico concept minimising possible risks in gas fermentations up-scaling. First, the economical feasibility of various fermentation methods is investigated. Then, two computational tools are presented using Clostridium ljungdahlii as model organism and synthesis gas as substrate in a 125 m3 bubble column reactor. The combination of economical investigation with modelling tools show high potential for successful scale-up of gas fermentations. With this concept feasibility, reactor design, operation mode and general risk minimisation can be analysed and specified.