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Title: High-throughput screening and biochemical characterization of human chromatin remodelers
Author: Chen, Y.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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This thesis describes the high-throughput screening of chrome/PHD domain constructs from a number of chromatin remodelers and subsequent biochemical and biophysical characterization of several constructs of human CHD3, CHD4 and CHD5. Results from NMR spectroscopy, size exclusion chromatography and small angle neutron scattering al indicate that these proteins are either misfolded, or that they form higher-order structures, and that this is likely caused by the praline rich/highly charged sequences conserved amongst the CHD3/4/5 double chromodomains. Pull-down experiments of three double PHD domain-containing constructs suggest that unmodified and methylated histone H3/H4 peptides bind to these proteins. A Drosophila homologue of human CHD3, dCHD3 which lacks the problematic sequences was then investigated. This expressed well but had low solubility. A refolding protocol was, however, successfully developed to recover dCHD3 from inclusion bodies. This protein was the only double chromodomain throughout the entire project that produced a high-quality NMR spectrum – in addition, it eluted from a gel filtration column as expected. Extensive crystallization trials were performed for all the constructs that could be produced in appreciable amounts and good purity. However, crystals formed only for the dCHD3 construct. High resolution reflections were observed, but the crystals proved not to be of the intact protein.  NMR structural studies of the human PHF8 PHD domain constructs are described – signals of 82% of the backbone residues were assigned with the missing residues most likely being unstructured.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available