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Serial and Single Crystal Studies of Ligand Binding to Radiation Sensitive Haem Protein DHP-B

Urban, Emily (2020) Serial and Single Crystal Studies of Ligand Binding to Radiation Sensitive Haem Protein DHP-B. Masters thesis, University of Essex.

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Abstract

Dehaloperoxidase (DHP) is a multifunctional globin and is the first known globin to have a biologically relevant peroxidase function. What makes DHP different from other globins is not just its peroxidase function, but its flexible distal histidine that is located at the opening of the distal pocket, this is essential to its peroxidase function. The main aims of this study are to better understand how and also where dehaloperoxidase binds its ligands, demonstrating different binding modes and pockets to better understand how it switches between its functions. Using both data collected during this project, and processing data from previous experiments, and comparisons of structures obtained from literature, binding modes of different ligands with the isoform dehaloperoxidase-B were explored. Another important question explored in this thesis, is the use of both room-temperature (serial) and cryogenic (single) data collection, to compare whether these two methods produce significantly different results. Single crystal data were obtained from Diamond Light Source (DLS) and Swiss Light Source, or from the protein data bank. Crystals were grown using the hanging-drop vapour diffusion method, soaked in cryoprotectant and measured at synchrotron sources. Serial data was collected using microcrystals at SACLA, Japan and from DLS. The structures were solved using both CCP4i2 suite and Phenix. The structures displayed different binding modes and pockets present in DHP-B, making bonds with multiple different amino acids present in the binding pocket. The distal histidine (His55) always plays a role in binding due its flexibility. The presence of a hemichrome species is also observed in almost all the structures in this study, including in room temperature, damage-free X-ray Free-Electron Laser (XFEL) structures, ruling out the possibility of the hemichrome species being from the use of cryoprotectant. More research needs to be done to determine whether the hemichrome species has any biological relevance. Comparisons of serial and single crystal studies do show some differences, indicating that temperature measured does have an effect on the structure.

Item Type: Thesis (Masters)
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry
Q Science > QR Microbiology
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Emily Urban
Date Deposited: 14 Jul 2020 15:47
Last Modified: 14 Jul 2020 15:47
URI: http://repository.essex.ac.uk/id/eprint/28164

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