The development of experimental and computational tools for studying protein interactions: Applied to the bacterial toxin pneumolysin

Rai, Nithin Sudharam (2001) The development of experimental and computational tools for studying protein interactions: Applied to the bacterial toxin pneumolysin. PhD thesis, University of Glasgow.

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Abstract

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

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Advisers: Tony Lawrence; Ron Poet; Olwyn Byron; Mattia Rocco
Keywords: Molecular biology, Bioinformatics
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-72697
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72697

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