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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

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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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

@phdthesis{Weyand2007Purif-8197, 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}, year={2007}, author={Weyand, Simone}, address={Konstanz}, school={Universität Konstanz} }

Weyand, Simone Reinigung, Kristallisation und Röntgenstrukturanalyse der Proteine des Lysinbiosyntheseweges von Mycobacterium Tuberculosis und Strukturanalyse von Membranproteinen von Deinococcus radiodurans R1 und Escherichia coli K12 Weyand, Simone 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.<br /><br />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?<br /><br />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.<br /><br />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.<br /><br />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. 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 2011-03-24T17:41:20Z deposit-license application/pdf eng 2007 2011-03-24T17:41:20Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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