Overcoming divalent cation sensitivity is not the only challenge for functional study of ABC transporters within polymer lipid particles

Abstract

Proteins of the ATP-binding cassette (ABC) transporter superfamily are involved in diverse biological processes including multidrug resistance. As membrane proteins, they exist within a complex lipid environment, and often it is necessary to isolate them from the other membrane components to study their structure, function, and dynamics. Traditionally, detergents have been used to isolate the transporters into micelles but this can strip away lipids that may be essential for function. Polymers such as styrene maleic acid (SMA) offer attractive alternatives to detergents as they retain the protein and lipids in a nanoscale disc. However, to date, no demonstration of full ABC transporter activity in these discs has been achieved, possibly due to the inherent divalent cation sensitivity of the SMA polymers; magnesium is essential for ATP binding to ABC transporters. Novel polymers such as those based on acrylic acid styrene (AASTY) show decreased sensitivity to divalent cations and, as such, may be well placed to probe ABC transporter activity. We have demonstrated that a range of commercially available AASTY polymers solubilise biological membranes efficiently, albeit with slightly different kinetics. ABC transporters can be solubilised and purified using AASTY polymers into discs of a comparable size to those formed by SMA2000. These discs show increased magnesium tolerance but, as for SMA2000, lipids within them do not seem to undergo a full phase transition. We were unable to detect ATPase activity of ABC transporters in AASTY polymer lipid particles, suggesting that magnesium tolerance alone is not sufficient to overcome the challenges.