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Title: Structural studies and molecular modelling of alpha-2u-globulin
Author: Adams, Paul David
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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It has been observed that certain small hydrocarbon molecules produce an increased rate of kidney damage (nephropathy) and incidence of renal carcinomas in male rats. Both d-limonene and 2,4,4-trimethylpentane are seen to be active nephropathic agents. Human exposure to these chemicals is widespread as d-limonene is used as a lemon flavouring in some lemonade drinks, and 2,4,4-trimethylpentane is a hydrocarbon from petroleum. The possible nephropathic effects of such chemicals in humans is cause of great concern. On the basis of biochemical research a model has been proposed for the mechanism by which these chemicals cause kidney damage in male rats. A non-covalent association between the chemical and the urinary protein alpha-2u-globulin (a2u) is suggested. This association reduces the susceptibility of the protein to lysosomal proteolysis. The subsequent acumulation of undigested protein in the lysosome eventually leads to cell death. Knowledge of the atomic-structure of the protein is needed to understand how these chemicals bind and how this binding could affect proteolytic sensitivity. The work presented attempts to determine this atomic structure by direct crystallographic methods and by indirect homology modelling with the known structures of related proteins. Crystals of a2u were grown, from protein purified to one molecular weight species, using the hanging drop method with ammonium sulphate as the precipitant. These crystals were small making X-ray analysis possible only with a synchrotron radiation source. The data collected indicated crystal twinning, which made interpretation of the data impossible. Efforts to improve crystal quality with narrower precipitant ranges and stabilising additives were unsuccessful. A homologous protein from mouse urine, major urinary protein (MUP), was purified using gel filtration followed by chromatofocusing and ion exchange chromatography. Crystallisation trials with this purified protein produced small crystals with the hanging drop method using either ammonium sulphate or ethanol as the precipitant. Crystals were both too small and temperature sensitive for X-ray analysis. Both a2u and MUP are members of a family of related proteins, the lipocalycins. The structures of three of the members of the family were available (human retinol binding protein, bovine beta lactoglobulin, and tobacco hornworm insecticyanin). It was possible to model the structure of a2u using these three crystallographically determined structures. Four different methods were used, the results of which were compared. The best model was selected on the basis of several criteria and was compared to the recently available structure of MUP. In all methods extensive energy minimisation in the presence of many solvent molecules was performed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available