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.

The study of protein structure and function is a fundamental step for understanding biological systems and for creating a solid ground for future drug development. Despite the development of novel ground-breaking biochemical and bioinformatical tools in the last decades, most proteins found in nature still have unknown assigned function. In this thesis, I present four publications aimed at protein structure and function determination. In these publications we present one method for high-throughput condition screening of membrane proteins for structural studies and three publications that aim at contributing to the understanding of proteins found in the pathogenic bacterium Mycobacterium tuberculosis, the deadliest bacterium worldwide, responsible for over 1 million deaths every year. Membrane proteins are very important targets for drug development. However, the study of membrane proteins is much more complex than fortheir soluble counterpart. In paper I, we developed a high-throughput method for detergent screening of membrane proteins that significantly reduces the time needed to find optimal conditions for structural studies. We developed amethod that allows for the screening of 192 protein-detergent conditions in 3 days. We used a TEV-folding reporter GFP-His tag vector to be able to measure the stability and amount of each sample after detergent solubilization of the cell lysate.In paper II, we characterized the Mycobacterium smegmatis respiratory supercomplex accessory proteins AscF and AscG. AscF and AscG arelocated in close proximity to the D and K proton pathways of Cytochrome c oxidase and might play a regulatory role in the respiratory supercomplex. Wes how how AscF and AscG do not bind nor metals nor nucleotides, but that they are essential for the normal growth of the M. smegmatis. We also show that both proteins are highly conserved in almost whole Mycobacteriales families, including M. tuberculosis. In paper III, we present two novel structures of the ferritin-like protein R2lox and further characterize the co-purified ligands in its lipid-binding pocket. We demonstrate how the two prevalent ligands that co-purify with the enzyme are fatty acids that host a C12 hydroxy-group and a double bond between the hydroxy group and the carboxylic group. The new structures from Sulfolobus acidocaldarius and Saccharopolyspora erythraea reveal new information about the enzyme’s possible regulatory mechanism and its possible interaction with partner proteins. In paper IV, we performed structural and functional studies of nicotinamide adenine dinucleotide (NAD⁺) synthase (NadE), which performs the last step of the de novo synthesis of NAD+: the amidation of nicotinic acidadenine dinucleotide (NaAD) into NAD⁺. We showed that some prokaryotes concert up to three NadE isoforms depending on external ammonium (NH³) availability. During high external concentrations of NH³, the ammonium-dependent NadE acts as primary catalyst of NAD⁺ synthesis. However, two glutamine-dependent homologues are capable of deamidation of glutamine when external ammonium availability is reduced. We show how the octameric glutamine-dependent NadE is the principal catalyst during nitrogen-fixing conditions, while the dimeric variant is the primary catalyst when NH³ availability is limited.