Principal Supervisor: Dr Alice RothnieLink opens in a new window

Co-supervisor: Alan Goddard & Ivana Milic

PhD project title: Investigating the effect of lipid environment on ABC transporter structure and function

University of Registration: University of Aston

Project outline:

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.

References:

Unger L, Ronco-Campaña A, Kitchen P, Bill RM & Rothnie AJ* (2021) Biological Insights from SMA-extracted proteins. Biochem. Soc. Trans 49(3):1349-1359 doi: 10.1042/BST20201067

Hardy D, Bill RM, Rothnie AJ* & Jawhari A* (2019) Stabilization of human multidrug resistance protein 4 (MRP4/ABCC4) using novel solubilization agents. SLAS Discovery 24; 1009-1017

Hardy D, Bill RM, Jawhari A* & Rothnie AJ* (2019) Functional expression of Multidrug Resistance Protein 4 (MRP4/ABCC4). SLAS Discovery 24; 1000-1008

Ayub H, Clare M, Milic I, Chmel NP, Böning H, Devitt A, Krey T, Bill RM & Rothnie AJ* (2020) CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups. BBA Biomembranes 1862; 183419

BBSRC Strategic Research Priority: Understanding the rules of life - Microbiology and Structural Biology

Techniques that will be undertaken during the project:

Protein expression (bacterial, insect cell, mammalian cell)

Membrane protein solubilisation (SMA or detergent)

Protein purification (affinity chromatography, SEC)

Fluorescence spectroscopy

Liposome formation and reconstitution

ATPase and transport assays

Mass spectrometry

Electron microscopy

 

Contact: Dr Alice RothnieLink opens in a new window