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Title: The development of experimental and computational tools for studying protein interactions, applied to the bacterial toxin pneumolysin
Author: Rai, Nithin Sudharam
ISNI:       0000 0001 3505 5165
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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Much attention has been given to the study of protein interactions, whether with other proteins (of the same or different type), with nucleic acids or with cell membranes. Such interactions can be understood by using structural models and by biophysical monitoring of the kinetics and structural basis of the interaction. Pneumolysin is a 53 kDa bacterial protein toxin that inserts into eukaryotic membranes where it self-associates to form pores in the membrane. This process involves the initial interaction between toxin and membrane, followed by interaction between toxin monomers. Because pneumolysin elicits an immune response, it also interacts with antibodies. It therefore represents a system that is involved in a variety of different interactions. The aim of this thesis was to develop modelling and experimental methodologies that would be used to further understand the complex behaviour of pneumolysin and its behaviour with other structures. The choice of methodologies were based on those used by Gilbert (1998) i.e. bead modelling and haemolytic studies. This thesis describes: the development and testing of the bead model generating program, SOMO; a purification protocol that used perfusion chromatography to purify pneumolysin, and an electrochemical instrument (Enzymometer) for monitoring haemolytic activity. SOMO was successfully tested on four experimentally characterised proteins: bovine pancreatic trypsin inhibitor, ribonuclease A, lysozyme and the much larger, dimeric proteia, citrate synthase. The results for the theoretical hydrodynamic predictions for these models show a very good correlation with experimental values particularly when water volumes of 24 A were used for modelling the hydration layer. This program is now ready to be tested on experimental systems such as pneumolysin. In preparation for this, pneumolysin was successfully purified (in a matter of minutes) by perfusion chromatography. While developing a continuous assaying method for pneumolysin using the enzymometer, it was shown that maximum toxin activity for both rabbit and sheep erythrocytes (at 37°C, in the presence of either magnesium or calcium divalent ions) occurred at approximately 0.4 mM Ca++ and at concentrations beyond 0.2 mM Mg++. Below these concentrations caused a decrease in toxin activity. Further studies also indicated a difference between ion leakage and haemoglobin leakage which suggests that cell lysis might occur by osmotic shock; a proposition thought unhltely based on previous studies (Blumenthal and Habig, 1984; Duncan, 1974).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral