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Title: NMR studies of the A domain of HMG1
Author: Hardman, C. H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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This thesis describes the solution structure of the A domain of rat HMG1. First, the A domain from rat HMG1 was subcloned, over-expressed and purified from E. coli. Initial characterisation revealed a mixed population of protein species resulting from both intra- and inter-molecular disulphide bond formation. To produce a suitable sample for NMR spectroscopy the non-conserved cysteine 22 was replaced with serine by site-directed mutagenesis. This mutant protein resembles the wild-type in binding to four-way junctions and short DNA duplexes. CD and NMR spectra suggest no major structural perturbations, and the protein remains fully reduced after many weeks in solution. The structure is very similar to that of the B domain. This together with the structure of Drosophila HMG-D protein (solved during this study), confirms that different HMG-boxes from the superfamily are likely to have the same global fold. Comparison with the recent structures of two different sequence-specific HMG-box:DNA complexes reveals both some similarities and differences. Some of the differences between the A domain and the other HMG structures were confirmed by studies of backbone dynamics of the protein (studied by others). The structure provides a framework to interpret many of the mutations generated in the HMG-box, and the starting point to discover the molecular differences responsible for the different properties of A and B boxes of HMG1 and between sequence and non-sequence specific HMG-box domains. The availability of isotopically labelled protein with full 1H, 13C and 15N assignments and known three-dimensional structure will provide the means to examine directly the nature of structure-specific DNA recognition by this novel class of protein using NMR spectroscopy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available