Using amphipathic polymers to understand membrane protein structure and function

Secondary Supervisor(s): Professor Alan Goddard

University of Registration: Aston University

BBSRC Research Themes:

• Renewable Resources and Clean Growth (Industrial Biotechnology)
• Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

Membrane proteins undertake vital roles in all cells and as a result are important pharmaceutical targets. To continue to develop effective therapies for diseases including cancer, neurodegeneration, and diabetes, and to address antimicrobial resistance, it is vital to develop a molecular-level understanding of what these proteins look like and how they work.

One challenge in this goal is the fact that membrane proteins have evolved to be embedded in lipid bilayers. There is a developing understanding that lipids are integral to the structures and functions of many of these proteins and their complexes. Approaches to the purification of membrane proteins increasingly focus on the replacement or, if possible, the retention of membrane lipids through the process. One such approach involves using amphipathic polymers to solubilise membranes into lipid/polymer particles which encapsulate proteins and their associated lipid bilayer.

The first generation of these polymers, styrene-maleic acids (SMAs) are remarkably effective at solubilising functional proteins from a broad range of biological membranes. However, they have three key limitations: their solubility in aqueous solutions is pH-dependent, sensitivity to divalent cations and size-limitations of the discs. Each of these can affect the ability of SMA to solubilise functional membrane proteins. A new generation of amphipathic polymers is under development that retains the useful properties of SMA but provide more flexibility to the method.

Two projects will be offered within this research area.

1. Understanding the assembly and structure of protein/lipid nanoparticle

Aims and objectives

1. 1. Characterise polymer-lipid particles containing different membrane proteins.

1. 1. Define key parameters for how protein-lipid particles assemble and behave in aqueous solutions.

1. 1. Develop improved methods for the purification of functional membrane protein complexes using amphipathic polymers.

2. Novel tools for tackling microbial resistance mediated by ABC proteins

Aims and objectives

1. 1. Optimise the purification of functional ABC proteins from Escherichia coli and/or Mycobacterial strains using novel amphipathic polymers.

1. 1. Use novel polymers to characterise the surface molecules present on pathogenic bacterial species.

1. 1. Produce protein samples for drug screening to identify mechanisms of multidrug resistance.

In each project the student will learn to express and purify membrane proteins in polymer/lipid particles and to analyse their protein and lipid content using a variety of biochemical and biophysical methods. These methods include native electrophoresis, drug-binding assays, small-angle X-ray scattering, fluorescence microscopy and electron microscopy. In one project we use a standard set of membrane proteins expressed in bacterial cells for method development work. In the other we would focus on ABC transporters from pathogenic species of bacteria (e.g. those causing MRSA and tuberculosis).

References

[1] Knowles TJ et al 2009: PMID: 19449872.

[2] Dörr, J.M. et al 2015: https://doi.org/10.1007/s00249-015-1093-y.

[3] Pollock NL et al 2019: https://doi.org/10.1016/j.bbamem.2019.05.011.