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Title: Technologies and methods to characterise Rubisco function
Author: Butt, Mohammed Salman
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Almost every carbon atom that our bodies are made of and clothed in, at one point or another saw the active site of the enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, one of the largest in nature at ~550 kDa, is also said to be the most abundant in nature, constituting up to 50% of soluble protein in land plants. It is however, notoriously inefficient at fixing carbon dioxide, due to its slow catalytic turnover, low affinity for atmospheric CO2, and its use of both CO2 and O2 as substrates for competing reactions. For this reason, Rubisco has also been one of the most intensively studied enzymes. The potential harvest yield of crops has been shown to be limited by the rate at which atmospheric carbon is fixed by Rubisco. As a result much focus is on studying and engineering Rubisco in order to increase the overall efficiency of biomass production, to cope with the fuel, fibre and food needs of a growing global population. Since Rubisco is one component of a system, the demand for developing tools to study and engineer Rubisco in light of its wider interactome and regulatory networks, is ever increasing. This thesis describes the development of tools to study the function of Rubisco. It includes the development of a novel method for purifying the enzyme using hydrophobic interaction chromatography. Different populations of Rubisco were isolated with distinct maximal rates of carboxylation, possibly reflecting differences in hydrophobic characteristics. It also contains the development of a spectroscopic activity assay for Rubisco activity, and an enzyme-linked immunosorbent assay (ELISA) for Rubisco and its catalytic chaperone, Rubisco Activase. These tools were then applied to investigate the influence of cellulose biosynthesis inhibition (CBI) on Rubisco in Arabidopsis seedlings, to investigate any potential sink-regulation of the activity or levels of Rubisco. It was found that specific Rubisco activity was reduced as a result of CBI, and rescued by providing osmotic support, thus implicating turgor pressure as a potential mechanism. Finally, preliminary results were obtained which demonstrate the potential of mass spectrometry for measuring relative levels of Rubisco ions generated by MALDI (matrix-assisted laser desorption ionisation), showing that this can be a useful method for use in interactome studies in conjunction with other tools.
Supervisor: Barter, Laura ; Klug, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available