Refine
Year of publication
- 2016 (3) (remove)
Document Type
- Doctoral Thesis (3)
Has Fulltext
- yes (3)
Is part of the Bibliography
- no (3)
Keywords
Institute
Cells perform a wide range of functions such as signalling, transportation, immunoprotection and metabolism. Unravelling the molecular mechanism behind those processes will provide a platform for more targeted and rational drug design. This is achieved by discerning the structural and functional aspects of the biological macromolecules involved. This thesis discusses about the biophysical characterization of protein structures and the biological importance of protein dynamics. Membrane receptors and enzymes which are ubiquitously present in our biological systems and regulate wide variety of functions are excellent choice for such study. From a pharmaceutical point of view, receptor and enzymes are exceptionally important drug targets as they represent the major share (receptor, 30% and enzymes, 47%) of all marketed drugs. Therefore, apart from biological insights, the detailed study of receptors and enzymes will provide the basis for new pharmaceutical applications. Most information about receptor activation and enzyme activity come from the structural and functional analysis of target members of the above mentioned systems.
In “Chapter 1 – General Introduction” the readers are introduced to the world of proteins with special focus on G-protein coupled receptors (GPCRs) and methyltransferases. The first part of this chapter discusses about GPCRs with emphasis on their classification, structural features and functions. GPCRs are the most abundant membrane receptors present in mammalian cells, accounting for almost 15% of all membrane proteins. The GPCR superfamily consists of ~800 members and can be subdivided into six classes (A-F). Class A containing rhodopsin, peptide hormones, olfactory GPCRs, is the most abundant with a large share of 85% of GPCR protein family. GPCRs share a common architecture of 7 transmembrane a-helices, with different ligand binding sites. Although a variety of ligands ranging from subatomic particles (a photon) to large proteins can activate a GPCR, their mechanism of signal transduction is almost similar. There are two major signal transduction pathways identified for GPCRs: the cAMP pathway and the phosphatidylinositol pathway. The therapeutic relevance of GPCRs has also been pointed out here since a large share (30%) of modern marketed drugs target GPCRs.
In the second part of this chapter, the structural and functional characterizations of methyltransferases (MTs) are discussed in detail. Several important biological processes in cells e.g. drug metabolism, gene transcription, epigenetic regulations are modulated by methylation of targets ranging from small biomolecules to large proteins. MTs are the proteins which catalyze this methylation reaction and transfer the methyl group to an acceptor molecule through SN2 like nucleophilic substitution reaction. The MTs can be classified on the basis of the substrate atoms they methylate: O (54% of all MTs), N (23%), C (18%), S (3%) and other acceptors (such as halides; 2%). They can also be categorized into five different classes (Class I-V) depending upon distinctive structural features facilitating substrate binding or catalytic activity. Rossmann fold and SET (acronym acquired from the Drosophila Su(var)3-9 and 'Enhancer of zeste' proteins) domain are the two characteristic structural motifs commonly found in MTs. Similar to GPCRs, MTs dysfunction has been shown to be involved in various diseases including neuropsychiatric diseases and cancer. Therefore they are also interesting targets for drug development. The final part of this chapter discusses the importance of structural biology in gathering information related to structure and conformational dynamics of proteins. The two prominent biophysical techniques used in structural biology, X-ray crystallography and NMR, are discussed with focus on their advantages and limitation. The importance of NMR spectroscopic techniques to investigate different dynamic processes of protein at atomic resolution under physiological conditions is also discussed. Real time NMR spectroscopy required for the analysis of slow protein dynamic processes (protein folding, enzyme catalysis, domain rearrangement) has been explained in detail.
The second part of the thesis (Chapters 3-4), which is the cumulative part, comprises the original publications grouped into 2 chapters according to their topic:
• NMR-spectroscopic characterization of the transiently populated photointermediates of bovine rhodopsin and it’s interaction with arrestin (Chapter 3)
• Structural and biophysical characterization of PaMTH1, a putative SAM dependent O-methyltransferase from filamentous fungi Podospora anserina (Chapter 4)
Each chapter is initiated by a detailed introduction to the topic, providing the framework for the following papers. The personal contribution of this thesis’ author to each publication is stated in the introduction to the respective article.
This thesis deals with the NMR characterization of the structure and the folding dynamics of DNA G quadruplexes as potential therapeutic target in cancer therapy and building block for DNA based nanotechnology.
The first part of this thesis (Chapters 1-5) introduces the reader to the world of G quadruplexes.
The main features of the classic Watson Crick double helix and alternative non B DNA structures are illustrated in Chapter 1. Many different base pairing schemes are possible, besides the canonical Watson Crick motif, thereby expanding the structural complexity of DNA. Non canonical base pairing, such as Hoogsteen hydrogen bonding, enables the assembly of triplets and quartets, which are the building blocks of triplex and quadruplex structures, respectively.
The structural characteristics of DNA G quadruplexes are delineated in detail in Chapter 2.
G quadruplex structures are extremely polymorphic, in terms of strands orientation, loops geometry, grooves width and arrangement of the glycosidic torsion angles. The various structural elements as well as the different cation coordination geometries are here presented, with a special emphasis on the diversity of conformations reported for the telomeric DNA G quadruplexes.
Chapter 3 describes the biological roles of G quadruplex structures in the genome. After introducing the architecture of the telomeric DNA and its interacting proteins, the mechanism of the telomeres elongation catalysed by the telomerase enzyme and its implications for cancer are discussed. The occurrence of G quadruplex structures in functional regions of the genome, such as promoter regions of oncogenes, and their possible roles in regulating the gene transcription are then outlined in the second part of the chapter.
The potential of G quadruplex as a novel anti cancer target is examined in Chapter 4 and the proposed anti cancer mechanisms for a ligand stabilizing G quadruplex structures are discussed.
RNA G quadruplexes and their putative role in gene regulation at the level of translation are briefly illustrated at the end of the chapter.
A general overview on the NMR methods to investigate the G quadruplex structures is presented in Chapter 5. The experimental set up used for the real time NMR studies of the G quadruplex folding is also described.
The second part of the thesis (Chapters 6-8), which is the cumulative part, comprises the original publications grouped in three Chapters according to the topic.
The state of the art on small molecules targeting G quadruplex structures is given at the beginning of Chapter 6, including a summary of the experimental structures of G quadruplexes in complex with ligands available up to date. The publications presented in Chapters 6.1-6.3 are concerned with the elucidation of the interaction modes between DNA G quadruplexes and selected ligands with potential therapeutic applications.
The binding ability of two natural alkaloids (berberine and sanguinarine) to telomeric G quadruplexes is examined in Chapter 6.1. The ability of carbazole and diguanosine derivatives (synthetized in the group of Prof. Dash, IISER, Kolkata) to interact with c-MYC G quadruplex and down regulate c-MYC expression is explored in Chapter 6.2 and Chapter 6.3, respectively.
The energy landscape of human telomeric G quadruplex structures is discussed in Chapter 7, in light of the experimental kinetic studies as well as molecular dynamics simulations reported in literature until now. Up to date there is no general consensus regarding the folding pathway of unimolecular human telomeric G quadruplex, in particular due to the lack of atomic resolution data on the species involved in the folding. Chapter 7.1 presents the first real time NMR study of the human telomeric G quadruplex folding kinetics.
The final chapter of this thesis (Chapter 8) outlines the potential of G-quadruplex structures as building blocks in nanotechnology. After illustrating briefly the additional possibilities offered by alternative non B DNA structures to programme nanomaterials, a number of applications employing G quadruplex structures in different fields of nanotechnology are described. The article presented in Chapter 8.1 investigates the structural and photoswitching properties of a novel intermolecular azobenzene containing G quadruplex synthetized in the group of Prof. Heckel (Goethe University, Frankfurt).
Fettsäuresynthasen vom Typ I (FAS I), hier bezeichnet als Fettsäuremegasynthasen,sind Multienzymkomplexe, in denen sämtliche funktionellen Domänen für die de-novo-Synthese von Fettsäuren einen strukturellen Verbund eingehen. Auch das für den Transport von Edukten und Intermediaten nötige Acyl Carrier Protein (ACP) ist kovalent gebundener Teil dieses Komplexes, der so zu einer hocheffizienten molekularen Maschine zur Massenproduktion dieser grundlegend essentiellen Zellbausteine wird. Die FAS I aus Pilzen (fFAS), als Gegenstand dieser Arbeit, mit einer Masse von bis zu 2,7 MDa ist heute in ihrer Struktur durch Röntgenkristallographische sowie elektronenmikroskopische Methoden gut charakterisiert. 48 funktionelle Domänen sind zu einem geschlossenen Reaktionskörper angeordnet, indem sie in einer strukturgebenden Matrix aus Expansionen und Insertionen bzgl. der enzymatischen Kerndomänen eingebettet sind, die 50% des gesamten Proteins ausmacht. Neben den zahlreichen strukturellen Informationen über fFAS ist jedoch noch wenig über ihre Assemblierung verstanden. Dabei ist sie nicht nur als ein Beispiel für das generelle Verständnis von Assemblierungsmechanismen von Multienzymkomplexen interessant, sondern wird hier auch als Ziel eines inhibitorischen Eingriffs betrachtet, um eine neue antimykotische Wirkstrategie abseits des Ausschaltens aktiver Zentren zu evaluieren. Nur wenn die Mechanismen und Wechselwirkungen im Assemblierungsprozess offen gelegt sind, lassen sie sich später gezielt attackieren. Essentielle Sekundärstrukturmotive müssen identifiziert und bewertet werden, um sie einer weiteren Evaluation als Drug-Target-Kandidaten zugänglich zu machen. In dieser Arbeit werden Resultate aus in-vivo-Experimenten an rational mutierten fFAS-Konstrukten unter Zuhilfenahme einer evolutionären Betrachtung der fFAS gemeinsam mit Erkenntnissen aus andernorts geleisteten in-vitro-Experimenten an fFAS-Fragmenten zu einem geordneten Assemblierungsweg der fFAS zusammengeführt. Dabei werden Evidenzen aus den Kausaltäten zentraler Anforderungen an einen Assemblierungsmechanismus der fFAS zu drei konsequenten Schlüsselschritten verdichtet, die (i) eine frühe Interaktion zweier komplementärer Polypeptidketten zu einer Pseudo-Einzelkette, (ii) eine posttranslationale Modifikation von ACP und (iii) die geordnete Reifung zum fertigen Komplex durch Selbstassemblierung der beteiligten Domänen umfassen. Durch rationale Mutationen an den Schnittstellenmotiven für die Pseudo-Einzelkettenbildung, werden diese als Schwachstelle der Assemblierung unterschiedlicher fFAS-Typen charakterisiert, wobei für S. cerevisiae nicht weniger als zwei gezielte Punktmutationen ausreichen, um die Assemblierung des gesamten Komplexes zu verhindern. Darüber hinaus zeigen Experimente mit fFAS-Konstrukten, deren Schnittstellenmotive einer intramolekular kompetitiven Wechselwirkung ausgesetzt sind, prinzipiell die Möglichkeit zur Inhibierung der fFAS-Assemblierung durch Störung der Pseudo-Einzelkettenbildung.