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Functional and structural characterisation of Mycobacterial Chaperonins

Ahmed, Mohammad Tabish (2010)
Ph.D. thesis, University of Birmingham.

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Abstract

In Mycobacterium tuberculosis there are 2 distinct groEL homologues which encode the chaperonins Cpn60.1 and Cpn60.2, with the latter predicted to be the main house-keeping chaperonin. Phylogenetic analysis has revealed that the genes for the duplicated chaperonins diverged a long time ago. This implies that the duplicated chaperonins have evolved for different cellular functions. Interestingly, while most chaperonins occur as stable large complexes with a characteristic double ring structure of 14 subunits, the Mycobacterial chaperonins are very unstable and appear to form much smaller complexes. Given that the large structures formed by most chaperonins are vital to their mechanism of action, it is unclear why the oligomers are so much less stable in Mycobacteria. In this study I present detailed functional and oligomeric analysis of the M. tuberculosis chaperonins. Using various biological techniques, including complementation assays, site directed mutagenesis, and domain swap experiments; I have been able to demonstrate that both chaperonins have evolved to fulfil alternate functions within the cell. I have shown that while Cpn60.2 is fully functional in E. coli, Cpn60.1 is not. However, M. tuberculosis Cpn60.1 was able to function in M. smegmatis and complement for the loss of its endogenous chaperonin. Domain swap experiments between Cpn60.2 and E. coli GroEL have provided further evidence to support the hypothesis that they function in a similar manner. Using analytical ultracentrifugation on purified proteins, I have been able to show that Cpn60.2 and its chimeric protein do form larger oligomeric complexes in vitro under certain conditions and in the presence of ATP.

Type of Work:Ph.D. thesis.
Supervisor(s):Lund, Peter A.
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Subjects:QR Microbiology
Institution:University of Birmingham
ID Code:1279
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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