In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from <i> Azotobactor vinelandii </i> complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. <p>In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources.

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Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 192-215).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 217-223).

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Polyarene dendrimers are composed entirely of aromatic rings and are of interest as they possess rigid and shape-persistent structures, which have potential applications in materials chemistry and as scaffold for functional molecules. This thesis describes the synthesis and characterisation of a number of new polyarene dendrimers containing pentaphenylbenzene-like (pentaaryl dendrimers) and hexaphenylbenzene-like (hexaaryl dendrimers) branching units which are capable of focal and peripheral expansions. These structures are based on terminal and internal 1,3-dialkyne cores, onto which two polyaryl sections were introduced via Diels-Alder reaction. These compounds possess inherent axial chirality as a result of the dialkyne position on the core, and a range of differently substituted polyaryl units which result in further chiral axes. Several crystal structures of pentaryl dendrimers were obtained, with these crystallising either as meso conformers or conformers with C2 symmetry. Introduction of a chiral auxiliary onto the central aromatic ring of these dendrimers provided evidence of the atropisomers in solution. These were studied via VT 13C NMR, revealing fast rotation and an energy barrier of about 66 kJ mol-1. The presence of additional aromatic rings on a hexaaryl dendrimer enabled observation of atropisomerism in solution owing to the greater steric hindrance of rotation of its branching units about the chiral axes. Fluorine-containing analogues of these dendrimers were synthesised and subjected to conformational studies through 19F-19F Exchange Spectroscopy (EXSY). Qualitative and quantitative analysis of the spectra revealed the kinetic and thermodynamic parameters, showing that the energy barrier of rotation of these dendrimers was within the range of 75-80 kJ mol-1, which was sufficient for spectroscopic observation but not for physical separation. Polyaryls with symmetry or lacking symmetry have been synthesised. The lack of symmetry was achieved by stepwise introduction of the polyaryl fragments. This demonstrates a route to polyaryls with sections possessing different structural features and thus potentially engineered properties and future chemical differentiations.

The work presented in my dissertation focuses on the conformational studies of bio-molecules including proteins and DNA using computational approaches. Conformational changes are important in numerous molecular bioprocesses such as recognition, transcription, replication and repair, etc. Proteins recognize specific DNA sequences and upon binding undergo partial or complete folding or partial unfolding in order to find the optimal conformational fit between molecules involved in the complex. In addition to sequence specific recognition, proteins are able to distinguish between subtle differences in local geometry and flexibility associated with DNA that may further affect their binding affinities. Experimental techniques provide high-resolution details to the static structures but the structural dynamics are often not accessible with these methods; but can be probed using computational tools. Various well-established molecular dynamics methods are used in this work to study differences in geometry and mechanical properties of specific systems under unmodified and modified conditions. Briefly, the studies of several protein and DNA systems investigated the importance of local interactions and modifications for the stability, geometry and mechanical properties using standard and enhanced molecular dynamics simulations. In addition to the conformational studies, the development of a new method for enhanced sampling of DNA step parameters and its application to DNA systems is discussed. Chapter 1 reviews the importance of the conformational changes in bioprocesses and the theory behind the computational methods used in this work. In the project presented in chapter 2 unbiased molecular dynamics and replica exchange molecular dynamics are employed to identify the specific local contacts within the inhibitory module of ETS-1. ETS-1 is a human transcription factor important for normal but also malignant cell growth. An increased concentration of this protein is related to a negative prognosis in many cancers. A part of the inhibitory module, inhibitory helix 1 (HI-1) is located on the site of the protein opposite to the DNA binding site and although loosely packed, stays folded in the apo state and unfolds upon ETS-1 binding to DNA. Our study investigated the character and importance of contacts between HI-1 and neighboring helices of the inhibitory module: HI-2 and H4. We also identified a mutant of HI-1, which possessed the higher helical propensity than the original construct. This study supported the experimental findings and enhanced the field by the identification of new potential target for experimental tests of the system, which plausibly inhibits binding to DNA. In the studies discussed in chapters 3-5 the conformational dynamics of DNA under normal conditions and upon specific epigenetic modifications are presented. Since DNA conformation can be accurately described by six base pair step parameters: twist, tilt, roll, shift, slide and rise, these were extensively analyzed and the results elucidated insights into the properties of the systems. In order to enhance unbiased simulations and allow for easier crossing of the energy barriers, we developed and implemented a novel method to control DNA base pair step parameters. With this approach we obtained the free energy estimates of e.g. DNA rearrangements in a more efficient manner. This advanced computational method, supported by standard and additional enhanced techniques, was then applied in the studies of DNA methylation on cytosine or adenine bases and oxidative damage of cytosine.

207 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Organic Chemistry, 1981

The present study exploits two different molecules as biorecognition elements for biosensing. In the first case, a protein biosensor was performed using maltose-binding protein (MBP). The ability to manipulate protein function rationally also offers the possibility of creating new proteins of biotechnological value. Our design has been used to test the understanding of allosteric transitions in proteins. Here we examined a simple conformational change that can represent the biorecognition principle for a reagentless biosensor. Previously, modular strategies for transducing ligand-binding events into fluorescent and electrochemical responses have been reported. Starting with a study of the conformational changes of MBP this research will further develop electrochemical maltose biosensors. The responses of four individual mutations (K46C-MBP-MT, N282C MBP-MT, Q72C-MBP-MT; and K25C-MBP-MT) were evaluated using square wave voltammetry. The possibility of using this type of transduction mechanism for sensor configurations and analyte specificity is discussed.The second part of this work involves SELEX (systematic evolution of ligands by exponential enrichment) and aptamers as biorecognition molecules. As a result of the SELEX method, we can obtain oligonucleotide sequences (aptamers) with recognition properties similar to antibodies. These synthetic elements play an important role in molecular recognition because of their capability for specifically binding of a target molecule. A new approach for the separation step has been performed, termed Soluble-SELEX. This new SELEX method uses hybridization as partitioning mechanism for separating the bound and unbound DNA members from the target-molecule. Hybridization procedure has been evaluated by fluorescence studies as partitioning mechanism for SELEX method. Herein, we exploited the incorporation of an aptamer for biosensing detection of a specific target molecule. Three different transduction methods such as fluorescence, electrochemistry and surface plasmon resonance (SPR) were evaluated. In all three cases, the biosensing procedure was successful.In conclusion, this research has evaluated the translation of a fluorescent biosensor into an electrochemical biosensor using maltose-binding protein as biorecognition element. On the other hand, a new SELEX method has been developed. However, future improvements are required in order to optimize the method. As result of SELEX a new avidin-aptamer was selected and three different transduction systems were employed to construct fluorescent, surface Plasmon resonance and electrochemical biosensors. / El presente estudio utiliza dos moléculas diferentes como elementos de bioreconocimiento. En el primer caso, un biosensor basado en proteínas fue desarrollado utilizando la proteína periplasmica de unión a maltosa (MBP = maltose-binding protein). La habilidad para manipular racionalmente la función de una proteína también ofrece la posibilidad de crear nuevas proteínas con valor biotecnológico. Nuestro diseño proteico ha sido usado para evaluar cambios alostéricos en proteínas. Este estudio evalúa un simple cambio conformacional el cual puede ser usado como el principio transductivo para un biosensor. Diferentes estrategias de transducción usando fluorescencia y electroquímica en eventos de reconocimiento entre la proteínas periplasmicas de unión y el ligando, han sido previamente reportadas. Esta investigación inicia con el estudio de los cambios conformacionales de MBP, continuando con el desarrollo de un biosensor electroquímico para maltosa. La señal de cuatro diferentes mutantes (K46C-MBP-MT, N282C MBP-MT, Q72C-MBP-MT; y K25C-MBP-MT) fue evaluada usando voltimetría de onda cuadrada. La posibilidad de usar este tipo de transducción mecánic (distancia) para la configuración de biosensores y la respectiva especificidad analítica es discutida. La segunda parte de este trabajo incluye el método SELEX (systematic evolution of ligands by exponential enrichment) y aptameros como moléculas de bioreconocimiento. Como resultado de el método SELEX, podemos obtener secuencias de oligonucleótidos (aptameros) con propiedades de reconocimiento similares a los anticuerpos. Estos elementos sintéticos, tienen un importante rol en el reconocimiento molecular por su capacidad de unión específica a la molécula blanco. Un nuevo mecanismo para el paso de separación ha sido realizado, y llamado SELEX-Soluble. Este nuevo método SELEX usa la hibridización como mecanismo de separación para dividir los oligonucleótidos de DNA que no se unen y los que se unen a la molécula blanco. El procedimiento de hibridización y su uso como mecanismo de separación en el método SELEX ha sido evaluado a través de estudios de fluorescencia. Este estudio también explora la incorporación de un aptamero como elemento de reconocimiento en un biosensor. Tres diferentes mecanismos de transducción has sido evaluados: fluorescencia, electroquímica y resonancia de plasmon superficial (SPR). En los tres casos una excelente señal fue reportada. En conclusión, esta investigación ha evaluado la transferencia de una biosensor de fluorescencia a un biosensor electroquímico, utilizando la proteína periplásmica de unión a maltosa como elemento de bioreconocimiento. De otro lado, un nuevo método SELEX ha sido desarrollado. Sin embargo, futuras mejoras son requeridas para optimizar el método. Como resultado del método SELEX realizado un nuevo aptamero para avidita ha sido seleccionado y tres diferentes sistemas de transducción ha sido empleado para construir tres diferentes biosensores (fluorescencia, electroquímica y SPR).