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Structural study of Phage lambda proteins

Al-Kaabawi, Naer (2018) Structural study of Phage lambda proteins. PhD thesis, University of Sheffield.

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Naer Alkaabawi final thesis in 26_3_2018b .pdf
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Lambda phage proteins Four tail proteins of the Lambda bacteriophage, Rap endonuclease, NinH, Ea31 and Ea10, have previously been expressed in E. coli. Here, the methods are reported that were used to optimize the expression, purification and crystallization of these proteins. The structure determination of Ea10 is described. No crystals have yet been obtained for the purified full-length Rap endonuclease. Therefore, two truncated versions of the Rap endonuclease, which removed the first 30 and 70 residues of the disordered N-terminus, were produced to potentially increase the chance of protein crystallization. Overexpression experiments for NinH and Ea31 proteins were carried out with a His- tag or an MBP-tag, respectively, for solubility reasons. Both proteins were purified and set up in many crystallization trials. Crystals were not observed. Although tag cleavage for these proteins was performed to increase possibilities of crystallization, It was difficult to obtain a suitable level of purified protein for setting up crystallization trials. Heavy-atom derivatized crystals were obtained for Ea10, from which its structure was solved by X-ray diffraction to 2.7 Å. It was found to form a dimeric structure with beta strands swapped between a beta sheet observed in each monomer. As yet, no strongly homologous structures have been found for Ea10 to help predict its main function. Structural comparisons for Ea10 via the Dali Server suggest a notable similarity with part of the Q-beta replicase core complex, indicating an association with RNA. Unpublished data from the laboratory of Dr. Gary Sharples (University of Durham, UK) suggests that it might have a DNA binding capability. However, assays described here have not been able to confirm this. Experiments are still ongoing to unravel the mystery of the Ea10 function. Structure determination for the full length DnaD protein of Bacillus subtilis, which is a 232 amino acid primosomal protein that binds to supercoiled forms of DNA and converts them to open forms without nicking, was attempted, involving the N- and C-terminal domains which have never been solved together. Both domain structures (N-terminal and C-terminal) were published independently using crystallography and NMR assay, respectively. High resolution crystals (1.6 Å) with different space groups were collected. These crystals were confirmed as the N-terminal domain hits. Despite the use of a protease inhibitor was used for the prevention of a protein cleavage, N-terminal crystals were formed. Protein-protein interaction assay with DnaA protein for stability increase has not given any progress.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Molecular Biology and Biotechnology (Sheffield)
Depositing User: Mr Naer Al-Kaabawi
Date Deposited: 03 Apr 2018 10:45
Last Modified: 03 Apr 2018 10:45
URI: http://etheses.whiterose.ac.uk/id/eprint/19904

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