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Title: Biochemical and biophysical characterisation of heat shock protein 90 and its domain interactions
Author: Rabu, Amir
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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This thesis describes the study of the Hsp90 proteins from Homo sapiens (Hsp90α) and Caenorhabditis elegans. The attempt to crystallise the C-terminal proteins is also described. The overall goal of the project was to biochemically and biophysically characterise various Hsp90 constructs and use this information to elucidate the biological role of this important family of proteins. The C-terminal domain of human Hsp90α. The work involved over-expression, purification and characterisation of the C-terminus of human Hsp90 α protein. The over-expression and purification led to the production of two reproducibly pure C-terminal human Hsp90 α protein constructs. The characterisation of these proteins focused on the interaction of the C-terminus with the immunophilin Cyp40 and also with ligands such as ATP and novobiocin. C. elegans Hsp90. The work involved the cloning, over-expression and purification of the N and C-terminus of C. elegans Hsp90.  The cloning of genes for the N and C-termini was successful. Purification of the proteins only led to the production of one C-terminal protein but no purified N-terminal protein could be obtained. Similar characterisation studies were carried out to the C-terminus of C. elegans Hsp90. As for the human C-terminal protein, the C. elegans C-terminal protein was found to bind to Cyp40 with a dissociation constant in the micromolar concentration range. The protein also binds to ATP and novobiocin with an affinity very similar to that of the C-terminal proteins of human Hsp90α. The C-terminus of C. elegans also exhibits ATPase activity but the activity was ten-fold lower than that of the C-terminal of human Hsp90α. The work presented in this thesis provides conclusive evidence of the existence of an ATP binding site in the C-terminal domains of the Hsp90 class of proteins. The biological relevance of this finding is also discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available