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

ABC（ATP结合盒）转运蛋白超家族存在于从细菌到人类的所有生物体中，它们利用ATP水解产生的能量为分子跨膜转运提供动力。人类转运蛋白参与广泛的功能，包括保护免受毒素，代谢，控制药物在体内的分布，介导炎症反应和转运脂质。一些成员负责遗传性疾病，如囊性纤维化和肾上腺脑白质营养不良，而其他成员则参与癌症治疗期间引起多药耐药性。众所周知，许多膜蛋白的功能受到其脂质双层环境的影响。特定的脂质可以直接与转运蛋白相互作用并调节功能，或简单地影响双层的一般性质（厚度，流动性）。本项目的目的是详细研究两种模型ABC转运蛋白的蛋白质：脂质关系，首先是细菌转运蛋白Atm 1，其次是人转运蛋白MRP 4/ABCC 4（多药耐药蛋白4）。Atm 1可以在大肠杆菌中容易地表达，使用苯乙烯马来酸聚合物（SMA）提取和纯化以形成SMA脂质颗粒（SMALPs）。我们之前已经证明了Atm 1可以从SMALP重组到脂质体中。在本项目中，将Atm 1复溶至具有规定脂质组成/性质的脂质体中，并监测对蛋白质功能的影响。这将与使用电子显微镜的结构研究相结合。MRP 4可以在Sf 9昆虫细胞或Freestyle HEK哺乳动物细胞中表达。将测量这些不同表达系统对蛋白质产量和功能的影响，并且SMA聚合物将用于从每个表达系统中提取和纯化MRP 4，以使得能够通过质谱法分析共提取的脂质。

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.