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X-ray crystallographic analysis of the archaeal transcriptional regulator TrmB and development of a graphical user interface for the monochromatic diffraction data processing software XDS

X-ray crystallographic analysis of the archaeal transcriptional regulator TrmB and development of a graphical user interface for the monochromatic diffraction data processing software XDS

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KRUG, Michael, 2009. X-ray crystallographic analysis of the archaeal transcriptional regulator TrmB and development of a graphical user interface for the monochromatic diffraction data processing software XDS [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Krug2009cryst-8832, title={X-ray crystallographic analysis of the archaeal transcriptional regulator TrmB and development of a graphical user interface for the monochromatic diffraction data processing software XDS}, year={2009}, author={Krug, Michael}, address={Konstanz}, school={Universität Konstanz} }

Krug, Michael X-ray crystallographic analysis of the archaeal transcriptional regulator TrmB and development of a graphical user interface for the monochromatic diffraction data processing software XDS Krug, Michael In 1970 Francis Crick published his "Central dogma of molecular biology", a framework for understanding the transfer of information between sequential information-carrying biopolymers,namely DNA, RNA and proteins. This idea occurred because all these biopolymers are linear polymers, so the sequence of their monomers can encode information. Transcription plays an important role in the flow of biological information because during transcription DNA information is copied into mRNA, of which subsequently proteins can be synthesized using the information in the mRNA as a template. But this linear flux of information is not always the same: like in big technical control loops there are several regulatory mechanisms that, depending on the information that is introduced into the system from diverse sources, control that flow of information. One such regulatory instance are transcriptional regulators. They control the information transfer from DNA to mRNA. TrmB from Thermococcus litoralis and Pyrococcus furiosus, two archaeal organisms, is such a transcriptional regulator. Gene expression in archaea relies on a eukaryotic-like transcription machinery and eukaryotic-like promoter elements but bacterial-like regulatory transcription factors. TrmB controls the expression of two different ABC transporters in P. furiosus, depending on the presence of different sugars (they are the substrates of the two transporters) within the cell. The DNA binding sites for TrmB in the two cases differ: one is palindromic, whereas the other is nonpalindromic. A big obstacle during this work was the low solubility of TrmB. This is why a truncated version lacking the DNA binding domain of the protein was constructed by Sung-Jae Lee: TrmB<sub>&#916;2&#8722;109</sub>. The structure of this sugar binding domain of TrmB with bound maltose could be solved at 1.5Å and led to an understanding of the sugar binding mode of TrmB. The sugar binding pocket of TrmB<sub>&#916;2&#8722;109</sub> does not resemble the canonical substrate binding pocket of eubacterial sugar-binding transcriptional regulators and periplasmic binding proteins and the bound maltose in TrmB<sub>&#916;2&#8722;109</sub> is sticking to the surface, wheras the sugars are bound to the eubacterial proteins deeply within the protein. Almost all hydrogen bonds between TrmB<sub>&#916;2&#8722;109</sub> and the maltose are formed between TrmB<sub>&#916;2&#8722;109</sub> and the nonreducing glucosyl residue of maltose whereas only one hydrogen is formed between TrmB<sub>&#916;2&#8722;109</sub> and the reducing glucosyl moiety of maltose. Extensive solubility tests with TrmB finally led to buffer conditions enabling to concentrate the protein to levels necessary for successful protein crystallization. Using this buffer, the protein could be crystallized with bound sucrose. The structure of TrmB revealed that the DNA binding domain is connected with the sugar binding domain via a short linker and consists of a winged helix-turn-helix motif preceded and succeeded by helices. The helices succeeding the winged helix-turn-helix motif of two monomers can form a coiled-coil arrangement leading to a TrmB dimer whose DNA binding domain architecture resembles those of other archaeal (putative) transcriptional regulators. This structure gives an idea of how TrmB could bind to its two different DNA binding sites. Another part of this work was the development of a Graphical User Interface for the data integration software XDS: XDSi. XDS is a text-based software for processing monochromatic diffraction data of protein crystals recorded by the rotation method. In the world of a crystallographer there are two big sticking points: producing diffracting protein crystals and determining the structure of the crystallized protein from the recorded reflection data sets. Like protein crystallization trials stand and fall with the purification protocol, determining the structure of a crystallized protein depends to a considerable part on the data integration step. The initial notion of XDSi was to have a Graphical User Interface to XDS that should automatically run XDS for a given dataset and subsequently produce plots representing the most informative data of the output files generated by the different XDS steps. In that way it would facilitate the handling of XDS for unexperienced users and save time for experienced users. The visualized output of important statistics would be easier to estimate and otherwise unrecognized errors could be avoided. XDSi is based on the scripting language Tcl in combination with the interpreter Tk (both implemented as C libraries) and the windowing shell wish. Since Tcl/Tk and the wish provide generic programming facilities as well as the ability to execute other programs, XDSi evolved into a Graphical User Interface that allows automatic processing and spacegroup assignment of one or multiple datasets so that the user can focus his finetuning efforts on the most promising of his datasets. application/pdf deu 2011-03-24T17:47:11Z deposit-license 2011-03-24T17:47:11Z 2009

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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