Williams, E.P. (2011) Structural and energetic characterisation of interactions at the nucleotide-binding site of Hsp90. Doctoral thesis, UCL (University College London).
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Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone linked to the maturation and activation of a wide range of 'client proteins'. These include steroid hormone receptors, receptor tyrosine kinases and p53. Hsp90 activates ‘clients’ through a series of conformational changes driven by the binding and hydrolysis of ATP in the N-terminal domain. These changes are thought to facilitate the formation of client protein binding sites and aid achieving their functional state. The aims of this thesis are to investigate the thermodynamics of nucleotide-based ligand binding to the N-terminal domain of Hsp90 and to study the mechanism by which conformational changes are induced by ligand binding. Thermodynamic characterization of ligand binding using isothermal titration calorimetry was carried out in order to investigate the contribution of the different groups of the nucleotide to the binding affinity and determined the importance of the charge state of the ligand to binding. Measurements of the change in constant pressure heat capacity induced by ligand binding were also performed in order to further investigate previously reported unusual heat capacity changes in this system that may correspond to differences in the conformational changes induced by ADP vs. an ATP analogue (AMPPNP) . Structural studies by NMR of the N-terminal domain reveal a high degree of flexibility and specific conformational changes in response to the binding of different ligands to the N-terminal domain of Hsp90. The thermodynamics of ligand binding to the full length protein and to the isolated N551 (the first 551 amino acids) were also investigated and found to have a significant influence over both the thermodynamics and the heat capacity measurements compared to the isolated N-terminal domain.
|Title:||Structural and energetic characterisation of interactions at the nucleotide-binding site of Hsp90|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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