Purification, crystallisation and X-ray structure analysis of proteins from the lysine biosynthetic pathway of Mycobacterium tuberculosis and structural studies of membrane proteins from Deinococcus radiodurans R1 and Escherichia coli K12
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Since the lysine biosynthetic pathway is present in bacteria, fungi and plants but absent from mammals its 9 annotated enzymes have drawn considerable attention as potential candidates for new drugs against tuberculosis. Within this work the 3 proteins Rv0858c, Rv1201c and Rv1293 have been selected for structural analysis. Rv0858c protein has been purified, crystallised and the structure determined to a resolution of 2.0 Å. In addition, pure protein of Rv1201c has been crystallised successfully and first attempts towards structure determination were carried out. For the third target, Rv1293, an existing dataset was used to re-build the model and to refine the structure. In addition this structure was compared in detail to the same target solved earlier in a different space group.
A detailed bioinformatical analysis was carried out for all 9 annotated proteins of the lysine biosynthetic pathway. The entire analysis was based on the questions: are too many or too few proteins annotated within the pathway?, is the order of proteins correctly annotated and is there any hint towards complex formation?
A second project dealt with the analysis of structures of several outer and inner membrane proteins. A new bacterial strain was therefore introduced to the lab and basic membrane protein laboratory procedures were established. Different well established protocols were applied in order to extract all the present membrane proteins from the outer membranes. Different candidate proteins were analysed by Maldi-tof mass spectroscopy and sequence comparisons.
Additionally the inner membrane protein Nramp of Deinococcus radiodurans R1 was selected for structural analysis. The target sequence of this protein was amplified by PCR, cloned into different vectors and the gene product overexpressed in different host organisms. The overexpression procedure was optimised and a purification protocol established. The protein was crystallised in lipidic cubic phases as well as by counter diffusion. These crystals were optimised and diffraction was obtained to a resolution of approximately 30 Å.
In order to set up a SAXS approach for membrane proteins, the model protein OmpF of E.coli was selected. The experimental set up was optimised and the shape of the obtained model reflected the high resolution structure which was clearly reproduced. Also, the Nramp transporter of the inner membrane was measured using this technique, but could not structurally characterised so far.
Zusammenfassung in einer weiteren Sprache
Da die Lysinbiosynthese nur in Bakterien, Pilzen und Pflanzen vorkommt, aber nicht in Säugetieren, sind die dort vorkommenden 9 annotierten Enzyme sehr interessant als mögliche Kandidaten für neue Anti-Tuberkulose Medikamente. In dieser Arbeit wurden die drei Proteine Rv0858c, Rv1201c und Rv1293 für Strukturanalysen ausgewählt. Rv0858c wurde gereinigt, kristallisiert und die Struktur bei einer Auflösung von 2.0 Å gelöst. Weiterhin wurde gereinigtes Protein von Rv1201c erfolgreich kristallisiert und erste Arbeiten zur Strukturbestimmung wurden ausgeführt. Der Datensatz für ein drittes Enzym Rv1293 wurde dafür verwendet, um ein Modell zu bauen und dieses zu verfeinern. Zusätzlich wurde die Struktur im Detail mit einer zuvor gelösten Struktur in einer anderen Raumgruppe verglichen.
Eine detaillierte bioinformatische Analyse wurde von allen annotierten Proteinen der Lysinbiosynthese durchgeführt. Die gesamte Analyse basierte auf den Fragen, ob zu viele oder zu wenige Proteine annotiert wurden, ob die Reihenfolge richtig ist und ob es Hinweise auf die Bildung von transienten Protein-Protein-Komplexen gibt.
Als zweites Projekt wurden Arbeiten in Richtung Strukturanalyse von äusseren und inneren Membranproteinen durchgeführt. Zunächst wurde hierfür ein neuer Bakterienstamm in das Labor eingeführt und die grundlegenden Techniken im Umgang mit Membranproteinen etabliert. Dafür wurden verschiedene hinreichend etablierte Protokolle angewendet, um die vorhandenen Membranproteine aus der äusseren Membran zu isolieren. Verschiedene überexprimierte Kandidatenproteine wurden mittels Maldi-tof Massenspektroskopie und Sequenzvergleichen analysiert.
Zusätzlich wurde das innere Membranprotein Nramp aus Deinococcus radiodurans R1 für die Strukturanalyse ausgewählt. Genomische DNA wurde amplifiziert und in verschiedene Vektoren kloniert. Das Genprodukt wurde dann in verschiedenen Bakterienstämmen überexprimiert. Die Überexprimierung wurde optimiert und ein Reinigungsprotokoll etabliert. Das Protein wurde kristallisiert und die Kristalle in einem Röntgenstreuexperiment untersucht. Streuung wurde bis zu einer Auflösung von ca. 30 Å beobachtet.
Um ein SAXS Experiment für Membranproteine aufzusetzen wurde das Modellprotein OmpF aus E.coli gewählt und dieser Prozedur unterzogen. Die experimentelle Durchführung wurde optimiert und die hoch aufgelöste Kristallstruktur weitestgehend reproduziert. Das Nramp Protein wurde anschliessend mit dieser Technik gemessen, konnte jedoch bisher nicht strukturell beschrieben werden.
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WEYAND, Simone, 2007. Purification, crystallisation and X-ray structure analysis of proteins from the lysine biosynthetic pathway of Mycobacterium tuberculosis and structural studies of membrane proteins from Deinococcus radiodurans R1 and Escherichia coli K12 [Dissertation]. Konstanz: University of KonstanzBibTex
@phdthesis{Weyand2007Purif-8197,
year={2007},
title={Purification, crystallisation and X-ray structure analysis of proteins from the lysine biosynthetic pathway of Mycobacterium tuberculosis and structural studies of membrane proteins from Deinococcus radiodurans R1 and Escherichia coli K12},
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