Polymer solubilisation and lipid characterisation of bacterial and fungal membranes

Thesis

Fungal infections are a major threat to immunocompromised patients. A potential target for treating such invasive infections is Pma1, a P-Type ATPase that pumps protons across the plasma membrane of fungi to create an electrochemical gradient essential for the secondary transport of nutrients. As yet, efforts to study Pma1 in vitro have been focused on detergent solubilisation and purification, which does not necessarily preserve the native lipid environment and can potentially alter the native hexameric assembly and ATPase activity of Pma1. This study had two main aims: (i) to develop a protocol for purifying Neurospora crassa Pma1 from its native source using polymer-lipid-protein particles, with the aim of maintaining a native lipid environment, and (ii) to characterise the native lipidome of the host organism N. crassa, and identify those lipid species that co-purify with Pma1. The effectiveness of several commercially available polymers in solubilising fungal membranes under various conditions were evaluated and compared to bacterial membranes. The well-characterised polymer, styrene maleic acid (SMA 3:1), was identified as a potential candidate for solubilising Pma1 from N. crassa, and limitations of this polymer for assessing protein purity and activity for fungal membranes were investigated. Using LC/MS to analyse the full N. crassa lipidome, 420 different lipid species were identified. By analysing lipids present in different purification steps of Pma1 in detergent, it was observed that purified N. crassa Pma1 hexamers are characterised by an enrichment of inositol-containing ceramides. Knowing these tightly associated lipid species will aid with future efforts to preserve Pma1’s native lipid environment during purification.

Authors: Cavalier, C

• DOI: 10.5287/ora-9ogok9jay
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: lipid-polymer nanoparticles; Pma1; membrane proteins; native environment; PMA; proton pump; DIBMA; SMA; lipid; lipidome; styrene-maleic acid copolymer; SMALP; lipodisq; structural biology; P-type ATPases; fungal membranes; lipidomics; neurospora crassa
• Subjects: Biochemistry; Lipidomics; Biophysics; membrane protein solubilisation; Lipids