Structural and Functional Studies of Membrane Proteins : From Characterisation of a Fatty Acyl-CoA Synthetase to the Discovery of Superoxide Oxidase

Abstract: This thesis is divided into three parts; the first part describes a method for efficient screening of membrane proteins for crystallography. By utilising the properties of a folding reporter GFP it is possible to quickly and accurately screen the stability of a protein in a range of conditions without full purification. This allows rapid assessment of the suitability of a protein for crystallography and a parallel optimisation of purification conditions for subsequent large-scale protein production.The second part describes the discovery of a membrane bound superoxide oxidase (SOO), a novel scavenger of membrane proximal superoxide. SOO is a kinetically perfect enzyme, reacting at rates close to the diffusion limit in a similar fashion to other superoxide scavengers, such as superoxide dismutase. We propose that SOO rescues electrons “lost” to superoxide and recycles them back into the respiratory chain, releasing oxygen. At the same time SOO contributes to the proton motive force by uptake of protons from the cytoplasmic side of the membrane.The third part concerns the fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis (M. tuberculosis). It represents a critical node point in M. tuberculosis lipid metabolism and has been suggested to be a vital component of M. tuberculosis survival in host cell macrophages. FadD13 harbours a hydrophobic cavity that is unable to house the very-long-chain substrates the enzyme has preference for. We propose that FadD13 is a peripheral membrane protein, utilising the membrane to house the very-long-chain fatty acid substrates during the activation reaction.