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Title: Development of an NOE driven method for obtaining robust and reliable models of large protein complexes
Author: Bruton, Shaun
ISNI:       0000 0004 5924 0551
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2015
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The majority of proteins interact with other proteins/nucleic acids to form functional complexes that are essential to the proteins cellular role. Solving the structures of these complexes is vital for a full understanding of a proteins function. However, many protein complexes have resisted attempts of structure determination by established methods, making modelling based on experimental data and known structures of individual proteins an attractive alternative. The work presented here describes the in silico testing, experimental validation and application of a technique that uses HN-CH3 NOEs to determine sequence-specific 13C/1H NMR assignments for side-chain methyl groups in proteins, which are generally abundant at protein-protein interfaces. The approach developed relies on the preparation of residually protonated protein samples, avoiding limitations imposed by the molecular weight of larger complexes. Using this approach it was possible to obtain comprehensive assignments for the methyl groups of IL-1β (17 kDa) both in the free form and in complex with a potential therapeutic Fab antibody fragment (a complex of 65 kDa). It was shown that these assignments could be used to identify a number of backbone amide to side chain methyl NOEs across the protein-protein interface. These NOEs provided a significant number of 1H-1H distance restraints that made a substantial difference to the accuracy and reliability of docked structures obtained for the IL-1β/Fab complex by restraint driven docking. This was confirmed by comparison to a crystal structure determined for the complex. The developed approach is both conceptually and experimentally straight-forward and is expected to be generally applicable to a wide range of protein complexes up to a molecular weight of approximately 100 kDa. The use of a homology model as the starting structure for the Fab fragment also demonstrates that this technique is tolerant of some differences in the starting and final structures.
Supervisor: Carr, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available