Investigating the effect of lipid environment on ABC transporter structure and function

The ABC (ATP Binding Cassette) transporter superfamily is found in all organisms from bacteria to humans, and they utilise energy from ATP hydrolysis to power the transport of molecules across a membrane. The human transporters are involved in a wide range of functions including protection from toxins, metabolism, controlling drug distribution in the body, mediating inflammatory responses and transporting lipids. Several members are responsible for genetic diseases, such as cystic fibrosis and adrenoleukodystrophy, whilst others are involved in causing multi-drug resistance during cancer treatment. It is well established that the function of many membrane proteins is affected by their lipid bilayer environment. Specific lipids may interact directly with a transporter and modulate function, or simply affect the general properties (thickness, fluidity) of the bilayer. The aim of this project is to investigate this protein:lipid relationship in detail for two model ABC transporter proteins, firstly a bacterial transporter, Atm1, and secondly the human transporter MRP4/ABCC4 (multidrug resistance protein 4). Atm1 can be easily expressed in E.coli, extracted and purified using styrene maleic acid polymer (SMA) to form SMA lipid particles (SMALPs). We have previously demonstrated Atm1 can be reconstituted from SMALPs into liposomes. In this project Atm1 will be reconstituted into liposomes of defined lipid composition/properties and the affect on protein function monitored. This will be combined with structural studies using electron microscopy. MRP4 can be expressed in either Sf9 insect cells or Freestyle HEK mammalian cells. The effects of these different expression systems on protein yield and function will be measured, and SMA polymer will be used to extract and purify MRP4 from each expression system to enable analysis of the co-extracted lipids by mass spectrometry.