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Title: Biophysical and crystallographic studies of immunophilin-ligand complexes
Author: Patterson, Alan F.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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The work in this thesis describes the cloning, expression purification and characterisation of human CypA and FKBP12 proteins. Various biophysical techniques were used to study and characterise the interactions of different ligands with these target proteins. Vectors containing the genes encoding each of these, for expression of both wild-type protein and protein with a hexa-histidine tag, have been cloned for both hCypA and hFKBP12. Crystals of hCypA have been grown, and the structures, in complex with a range of small molecules including dipeptides, and dimedone derived ligands, have been solved. hCypA crystal complexes were also used to further explore the nature of the ‘crystal dissociation constant, Kcryst’ using the depetide ligand Val-Pro. Crystals were soaked in various ligand concentrations and by solving the X-ray structures of these complexes, ligand occupancies could be determined providing an experimental value for the equilibrium binding constant, Kcryst = 23mM. This compares well with a Ki value 19mM measured using an enzyme inhibition assay. Protein-ligand binding data measured using a range of methods is also presented, including fluorescence spectroscopy, mass spectrometry and surface plasmon resonance. It has also been demonstrated how the technique of surface plasmon resonance may be optimised to provide a sensitive ligand-binding assay. By tethering protein to a Ni2+ chip via an N-terminal hexa-his tag followed by covalent linking via primary amines, a stable and active surface was obtained. This allowed a competition binding assay to be developed, which allows the binding of ligands of Mr 250-500Da to be measured. The work done here should help further the design of potential inhibitors of hCypA, and in the measurement of their binding affinities; and possibly aid in the future design of anti HIV-1 therapies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available