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Title: NMR studies of DNA and RNA binding proteins
Author: Hill, G. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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HMG-D is a 112-residue, non-histone chromosomal protein from Drosophila melanogaster and is a member of the class of non-sequence specific HMGB proteins. The present project was based on the observation that other HMGB complexes that had been solved by NMR had a phenylalanine residue at a key interfacial location (corresponding to position 12 in HMG-D), whereas those like HMG-D that gave few intermolecular NOE cross peaks generally had a tyrosine at this location. This tyrosine was known to be involved in hydrogen-bonding to the DNA in a related complex that had been solved crystallographically. The Y12F mutant of full-length HMG-D was expressed and purified in isotope-labelled form suitable for NMR spectroscopy, and a set of multidimensional triple resonance experiments used to derive assignments for the backbond resonances of the protein both free and in complex with the dA2 bulge DNA. Sidechain assignments for the protein were obtained by a combination of “CCH”-transfer-based experiments and NOE spectra, while nearly complete assignments for the DNA in the complex were obtained from a combination of homonuclear 2D NOESY and TOCSY experiments together with filtered NOESY experiments where just cross peaks between protons both of which were not coupled to heteronuclei were selected. Filtered NOESY-based experiments were used to observe intermolecular NOE cross peaks in isolation, and, in contrast to the case of the wild-type complex, these experiments yielded around 50 intermolecular interactions. Together with an extensive set of assigned intramolecular NOE constraints, these formed the basis for a calculation of the structure of the complex starting from random conformations of both protein and DNA chains, which resulted in an NMR structure for the complex that had good precision over the structured region (residues 3-70 of the protein and stem 1 of the DNA).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available