SMALPs: breaking the barrier to study ABCB1 in its native lipid environment
- Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia.
- School of Life Sciences, University of Nottingham, Queens Medical Centre, University of Nottingham, Nottingham, UK.
The multidrug resistance P-glycoprotein (ABCB1) has ability to bind and extrude a vast array of chemotherapeutic drugs out of cancer cells. ABCB1 has four pharmacologically distinct drug binding sites and recent work by our laboratory has provided preliminary locations for them . The ongoing aim of our research is to provide detailed locations of the binding sites and their physical properties. Three of the drug binding sites are located at the protein-lipid interface and it is clear that the lipid environment has a significant influence on drug binding. Consequently, we intend to use the extraction procedure using styrene malic acid (SMA) co-polymer. SMA makes nanodiscs, termed as Styrene Malic Acid Lipid Particles (SMALPs). A key feature of SMALPs is their ability to encapsulate membrane proteins along with native lipids, particularly those at the annular region. SMALP based extraction has been compared with conventional method using the detergent dodecyl-maltoside based (DDM). The two methods were compared in terms of ABCB1 solubilisation efficiency, yield, purity and homogeneity at different stages during the purification procedure. DDM was more effective at solubilisation of ABCB1 and displayed higher affinity binding to the Ni-NTA chromatographic resin. Overall, the final purity between the two preparation procedures did not differ. A considerable advantage of SMALP based extraction is the ease of centrifugal concentration, with minimal losses compared to the DDM-solubilised protein. Finally, the final yield of purified protein did not differ between the two procedures. The SMALP system has been optimised for preparation of ABCB1 at high concentration with the crucial native lipid environment maintained.  R. Mittra, M. Pavy, N. Subramanian, A.M. George, M.L. O’Mara, I.D. Kerr, R. Callaghan, Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein, Biochem Pharmacol 123 (2017) 19-28.