The mycobacterial membrane protein large 3 (MmpL3) is responsible for the transport of key precursor lipids to the periplasm of Mycobacteria. Multiple structures of MmpL3 from Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msmeg) in various conformational states indicate that the protein is structurally and functionally monomeric 1–4. However, all other resistance, nodulation and cell division transporters structurally characterised to date are either dimeric or trimeric 5. In this thesis, an in-depth biophysical and computational analysis of the oligomeric status of Msmeg and Mtb MmpL3 was carried out. MmpL3 was extracted from the membrane either into styrene maleic acid lipid particles (SMALPs) or purified in detergent and reconstituted into synthetic nanodiscs. Initial evidence on migration pattern in native and denaturing gels, as well as Western blot analyses showed the presence of two distinct MmpL3 populations, likely monomeric and dimeric protein. Analytical ultracentrifugation experiments on size exclusion chromatography fractions confirmed that the protein not only exists as a monomer but also arranges into homodimers, both in SMALPs and synthetic nanodiscs. Furthermore, sucrose gradient separation of MmpL3 populations from Msmeg reconstituted into synthetic nanodiscs allowed clear identification of the presence of both protein arrangements using laser induced liquid bead ion desorption (LILBID), a native mass spectrometry technique. Preliminary cryogenic electron microscopy analysis on the same sample confirmed that the protein forms physiological dimers in nanodisc particles. Untargeted lipidomics experiments on MmpL3 co-purified lipids revealed phosphatidylethanolamine and phosphatidylglycerol as the predominant lipid classes. Therefore, Molecular Dynamics simulations were performed on a dimer model embedded in a physiologically relevant lipid composition, revealing the potential protein-protein interface and suggesting that lipids may play a role in stabilising the dimeric arrangement. In conclusion, all results presented in this work show that Msmeg and Mtb MmpL3 can exist as homodimers in a variety of membrane mimetic systems.