Investigation into the basis of protein-lipid interactions for ATP-Binding (ABC) transporters, using novel polymer-based solubilisation

Abstract

ABC transporters such as BmrA are key targets for structural and functional characterisation due to their role in multi-drug efflux and the resulting development of multi-drug resistance. While a wealth of knowledge is available regarding the structure of some of these membrane proteins thanks to developments in techniques such as cryo-EM, functional study remains limited. Using the traditional detergent solubilisation method, concerns are raised regarding the stability and integrity of studied membrane proteins due to the non-native nature of detergent micelles. Due to this, novel alternatives such as amphipols, membrane scaffold proteins and co-polymers were introduced, but each method displays their own advantages and disadvantages. Solubilisation of membrane proteins using co-polymers has been studied here as they have the unique ability to retain the native phospholipid environment resulting in the production of highly stable nanodiscs with intact protein-lipid interactions. However, co-polymers such as SMA2000 display a sensitivity to divalent cations which is a concern for ABC transporter study due to the requirement of magnesium as a co-factor for ATPase activity. To overcome this issue, a range of co-polymers have been tested in the solubilisation of ABC transporters BmrA and MRP4 in bacterial and insect membrane models. In this project, SMA variants and Glyco-DIBMA were relatively unsuccessful at solubilising BmrA. However, DIBMA and AASTY co-polymers solubilised BmrA at a good purity and yield with nanodiscs displaying a lower sensitivity to magnesium. Further experimentation found BmrA to be capable of substrate binding in selected co-polymer nanodiscs, but while SMA2000 and DIBMA solubilised BmrA did not display ATPase activity, 6-50 and 11-50 solubilised BmrA did. Nanodiscs characteristics such as size, polydispersity and lipid composition were also investigated. DIBMA appears to produce larger nanodiscs with a wider distribution when compared to SMA2000, 6-50 and 11-50, but all nanodiscs displayed an enrichment of PE when compared to crude membranes. Co-polymer solubilisation has been shown to be effective in the study of membrane proteins, with characterisation in this project finding AASTY 6-50 and 11-50 to be superior polymers for the functional study of BmrA.