1,721,009 research outputs found
Qualitative and quantitative characterization of protein backbone heterogeneity by solid-state NMR spectroscopy
Full text linkFlexibility 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
Structure and dynamics studies of the enzymes hCAII and GlpG via NMR spectroscopy
No full textLösungs- 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.Solution and solid-state NMR are valuable tools to characterize the chemical environments and dynamics of molecules like proteins with atomic resolution. Here, the effects of protein-ligand interactions on structural features and flexibility, strategies for NMR sample optimization, as well as different tools for peak assignments were elucidated.
For an unlabelled ligand in hCAII-bound state, dynamics were detected for a high-affinity complex via dedicated solution and solid-state NMR experiments. The enzyme hCAII is a well-studied drug target involved in, for example, pH regulation in human cells. The approach allowed to connect protein-ligand interactions with ligand flexibility, which might be of interest for medicinal chemistry. Furthermore, time-shared 3D NOESY/RFDR experiments were utilized both in solution and the solid state to assign methyl groups of hCAII.
Intra-membrane proteases of the rhomboid-like family are involved in processes associated with devastating conditions like cancer and neurodegeneration[1]. Here, GlpG from E. coli as a well-studied representative of the family was studied. After optimizing several expression and purification conditions, selected methyl groups were assigned via mutation to study chemical shift perturbations and microsecond to millisecond motion via solution NMR spectroscopy, comparing GlpG in apo and inhibitor-bound state. Our data indicate that upon binding of inhibitor STS1775, the conformational ensemble changes despite of up to 18 Å distance from the active site. Consequently, the occupancy of the active site modulates conformations close to but also far away from the substrate binding site
Characterisation of protein structure and dynamics by NMR spectroscopy and computational methods
No full textNuclear 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
Deciphering strand-asymmetrically modified CpG dyads in the DNA double-helix
No full textAlle Lebewesen müssen dafür Sorge tragen, in ihrem umfangreichen Erbgut die gerade für sie überlebensnotwendigen Gene von denen zu unterscheiden, die nicht gebraucht werden. Insbesondere mehrzellige Organismen müssen diesen Prozess zellspezifisch koordinieren, trotz dessen, dass hier dieselbe Erbinformation in allen Zellen des Individuums vorliegt. Ein Mechanismus, welcher diesem Zwecke dient, ist die Modifikation von DNA-Nukleobasen, den Bausteinen des Trägers der Erbinformation.
In Säugetieren wie Mensch und Maus kommt hierbei der Methylierung der DNA-Nukleobase Cytosin am Kohlenstoffatom C5 des Pyrimidinrings eine besondere Rolle zu. Sie findet auf beiden Strängen der DNA-Doppelhelix innerhalb des kurzen Sequenzpalindroms CpG statt und trägt entscheidend dazu bei, dass hier ortsspezifisch andere molekulare Interaktionen für die Expression der Erbinformationen notwendig werden. Da das Produkt 5-Methylcytosin für weitere enzymatische Modifikationen wie der Oxidation zu 5-Hydroxymethylcytosin, 5-Formylcytosin oder 5-Carboxycytosin zur Verfügung steht, können unterschiedliche Kombinationen dieser Cytosinderivate mit gänzlich einzigartigen chemischen Eigenschaften an den komplementären CpG-Paaren im DNA-Doppelstrang vorliegen. Ein Aspekt, der unter dem Gesichtspunkt der epigenetischen Funktion dieser Derivate in Ermangelung technologischer Innovation sie in natürlichem Chromatin zu untersuchen, bislang kaum erschlossen werden konnte. Inwiefern es nun möglich ist, solche Strang-symmetrischen oder Strang-asymmetrischen Kombinationen von Cytosinderivaten in diesen CpG-Paaren auf molekularer Ebene in der DNA-Doppelhelix zu erkennen und somit gegebenenfalls zu entschlüsseln, ist Gegenstand der vorliegenden Arbeit.
Ausgehend von verschiedenen Homologen einer Proteindomäne, welche symmetrisch methylierte CpG-Paare erkennen, den Methyl-CpG-bindenden Domänen (MBD), wurden aufgrund struktureller Erwägungen und funktionaler Studien der MBD–DNA-Binding, degenerierte Proteinvariantenbibliotheken erstellt. Mithilfe eines hierfür eigens entwickelten Hochdurchsatzverfahrens gelang es, Varianten zu identifizieren, die nahezu selektiv eine aus fünfzehn Paarungen obiger Cytosinderivate im DNA-Doppelstrang erkennen. Neben allgemeinen Substitutionsprofilen für verschiedene Paarungen wurden im Speziellen mehrere MBD-Varianten entdeckt, die eine neue, natürlicherweise nicht vorhandene Selektivität für 5-Hydroxymethyl- und 5-Carboxymethylcytosin-haltige CpG-Paarungen aufwiesen. Aus der weiteren biochemischen und strukturellen Charakterisierung der Bindespezifität konnten einige Erkenntnisse über die molekulare Erkennung Strang-asymmetrisch modifizierter CpG-Paarungen gewonnen werden, welche in Zukunft als Schlüssel dienen können, die epigentische Funktion der Cytosinmodifizierung im humanen Genom mithilfe solcher speziell auf sie zugeschnittenen Sonden zu entschlüsseln
Makes caterpillars floppy toxins Mcf1 and Mcf2 from Photorhabdus luminescens
No full textThe Makes caterpillars floppy toxins Mcf1 and Mcf2 are essential virulence factors from Photorhabdus luminescens, that ensure the survival of the bacterium and its symbiotic nematodes. In my thesis I solved the structure of Mcf1 (3.6 Å) and described Mcf2 as more instable and flexible, which prevented high resolution structure determination but still gave rise to a partial cryo-EM density. Both toxins consist of an N-terminal effector domain (NED), activator binding domain (ABD), protease effector domain (PED), two putative translocation domains (TD) and three putative receptor-binding domains (RBD). While the general structure resembles the ABCD toxin family, the translocation domains in particular are similar to the translocase of large clostridial toxins (LCT). Finally, I identified Arf3 as the cytosolic activator of both toxins and could formulate an activation mechanism that causes the release of the NED and PED into the cytosol, where both act independently as toxic effectors that contribute to the high potency of the Mcf toxins
Characterization of protein structure and dynamics by solution and solid-state NMR
No full textThe 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
Optochemical control of DNA methylation erasers and readers enables kinetic insights into their domain-dependent interplay
Full text link5-Methylcytosine (5mC) is a central epigenetic mark of mammalian DNA. It mainly occurs in cytosine-guanine (CpG) dinucleotides and is recognized competitively by methyl-CpG binding domain (MBD) proteins and ten-eleven-translocation (TET) dioxygenases, which act as methylation readers and erasers to mediate regulatory chromatin crosstalk and epigenome editing, respectively. The dynamic reader-eraser interplay at their common substrate is therefore highly regulated for a coherent transcriptional program. However, mechanistic insights of their interplay are hampered by a lack of suitable methodology to control their activities in living cells.
This work employs light-activatable human TET1 and MBD1 to enable precise temporal control of enzymatic oxidation activity or substrate recognition. Light activation is achieved by genetic encoding of a photocaged serine that can be co-translationally incorporated at critical protein sited in mammalian cells. On the one hand, monitoring the TET1-catalyzed 5mC oxidation kinetics in vivo reveals a multifaceted domain-dependent modulation by MBD1. While the MBD domain of MBD1 negatively regulates TET1 oxidation kinetics and dominates the interplay by competing for the 5mC substrates, the third Cys-x-x-Cys (CXXC3) domain of MBD1 contrarily modulates TET1 activity by binding to nonmethylated CpGs. Intriguingly, the transcriptional repressor domain (TRD) does not influence 5mC oxidation kinetics by TET1. On the other hand, studies with light-activatable MBD1 indicate a domain-dependency of cellular mCpG binding kinetics. Depriving the nonmethylated CpG affinity of the CXXC3 domain enhances binding kinetics, whereas the absence of the TRD domain results in decreased binding kinetics. Moreover, the light-activatable MBD1 can further unveil the mechanism of MBD1-TET1 interplay by uncoupling the process from prior binding events of MBD1. Collectively, this work enables first kinetic insights into the domain-dependent interplay of methylation readers and erasers in the natural chromatin context and provides novel tools to unravel the dynamic chromatin regulation program
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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