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Title: Structure determination of protein-ligand complexes using 1H-NMR chemical shift changes
Author: Cioffi, Marina
ISNI:       0000 0001 3551 7163
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2007
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Chapter 1 reviews the experimental and computational techniques available for the structure determination of protein-ligand complexes at atomic resolution. Chapter 2 describes a new method for determining three-dimensional solution structures of protein-ligand complexes using experimentally determined complexation-induced changes in ¹H NMR chemical shift (CIS). The method has been tested using the complex formed by the protein Neocarzinostatin and four synthetic chromophore analogues. The experimental CIS values were used in a continuous direct structure refinement process based on genetic algorithms to sample conformational space. The calculated structure agrees well with the NMR solution structure of the complex indicating the potential of this approach for structure determination. Chapter 3 reports an analysis of the effects of protein flexibility on the accuracy of 3D structures determined using the CIS-based approach. The effects of protein conformational mobility have been investigated. The results indicated that loop movement has a significant impact on the quality of the structure generated by the CIS structure determination methodology. Chapter 4 introduces a first attempt to validate the CIS-based approach on a set of complexes containing ligands, with a range of anisotropic functional groups, molecular size and flexibility. For all the cases analyzed, it was possible to identify the protein binding site and in some cases the orientation of the ligand in the binding site was also accurately predicted. Chapter 5 reports binding studies for the complexes of the protein Barnase with ligand 3-GMP, d(GpC) and d(CGAC). The mononucleotide did not show any binding even when changing pH conditions. However, binding was observed for the dinucleotide and tetranucleotide. NMR titration experiments supported by structure determination experiments show that in both cases the ligand binds in the G-recognition binding site, in contrast with previously published X-ray experiments, where d(GpC) tends to occupy secondary subsite as a result of crystal packing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available