X-ray crystallographic analyses of archaeal DNA binding proteins TrmBL2 and TrmB


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AHMAD, Misbha Ud Din, 2015. X-ray crystallographic analyses of archaeal DNA binding proteins TrmBL2 and TrmB

@phdthesis{Ahmad2015X-ray-32241, title={X-ray crystallographic analyses of archaeal DNA binding proteins TrmBL2 and TrmB}, year={2015}, author={Ahmad, Misbha Ud Din}, address={Konstanz}, school={Universität Konstanz} }

X-ray crystallographic analyses of archaeal DNA binding proteins TrmBL2 and TrmB 2015-11-25T09:17:23Z 2015-11-25T09:17:23Z Ahmad, Misbha Ud Din eng In archaea, packaging of DNA into chromatin and transcriptional regulation are closely related processes and the proteins involved therein often exhibit overlapping roles. In case of extremophilic archaea, the challenge to protect the DNA from detrimental effects of the harsh environments has led many of these chromatin proteins to evolve the additional function of DNA protection. The multiple roles these proteins play in DNA metabolism makes them interesting candidates for structural studies.<br />The first and major part of this work deals with the structural elucidation of TrmBL2, a member of the TrmB family of transcriptional regulators. Recent studies have to a great extent established the role of TrmBL2 in both chromatin shaping and transcriptional regulation. Upon association with DNA, TrmBL2 has been shown to form thick fibrous structures. Deletion of TrmBL2, in addition to the loss of fibrous structure, leads to upregulation of many unrelated genes thereby providing evidence for its role as a dual functional protein.<br />For structure determination, TrmBL2 was heterologously expressed in E.coli, subjected to ion-exchange and size exclusion chromatographic purification and crystallized with 19 or 17 bp TGM (Thermococcales Glycolytic Motif) dsDNA. In the absence of a suitable molecular replacement model, phases were determined by the Selenium Single Wavelength Anomalous Dispersion (Se-SAD) method. The structure of DNA-free TrmBL2 expressed in Pyrococcus furiosus was subsequently determined by molecular replacement.<br />TrmBL2 crystallizes as a tetramer in an asymmetric unit, both in the DNA-bound and DNA-free forms. The structure reveals an extended winged Helix Turn Helix (ewHTH) domain at the N-terminus followed by a coiled coil dimerization domain and a Phospholipase D (PLD) like domain at the C-terminus. While the electron density of the sugar-phosphate backbone of the bound TGM dsDNA is clearly distinguishable, the density for the nucleobases is averaged and represents a superposition of three binding modes with a 3 bp shift around the central 19 bp DNA at the 5’ and 3’ ends thereby explaining the observed 25 bp density. For the 17 bp DNA, the observed 21 bp density could be explained by a similar 2 bp shift at the 5’ and 3’ ends. During refinement, the occupancy of the nucleotides was adjusted so that the overall occupancy sums to the actual number of the base pairs used in crystallization. Given the non-specific binding of TrmBL2 to the DNA, the observed multiple binding modes and the resultant averaging out of the nucleobase density is not surprising.<br />The structure of the DNA-free TrmBL2 does not show any major differences from the DNA-bound structure.<br />The TrmBL2-DNA complex structure described in this work shows a hitherto unknown mode of tetramerization and DNA binding. The analyses of the crystal structures provide a basis for the reported non-specific binding of TrmBL2 to the DNA and also provides an explanation for its observed roles in chromatin structuring and transcriptional regulation.<br />The second part of this work details the efforts to devise a protocol for overexpressing TrmB and to overcome the low solubility issues of this protein with the ultimate aim of solving the structure of TrmB in complex with TM and MD promoters. Towards this end several constructs were tried but the problem of TrmB proteolysis proved to be a major hindrance in the realization of these goals. Ahmad, Misbha Ud Din 2015

Dateiabrufe seit 25.11.2015 (Informationen über die Zugriffsstatistik)

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