Understanding and improving SMALP solubilisation and purification of membrane proteins

Project Outline

Membrane proteins play a wide range of important roles in the cell, including controlling what enters and leaves cells and mediating cellular communication. This makes them potential drug targets for many conditions. However, due to their location within the lipid bilayer membrane, it is more challenging to study their structure and function than soluble proteins. Traditionally detergents were used to solubilise membrane proteins by disrupting the lipids and forming a micelle around the hydrophobic portions of the membrane protein. However, the detergent micelles do not fully mimic the environment within the bilayer, and this can be detrimental to the protein structure or stability. In 2009 a different approach was reported, using the co-polymer styrene maleic acid (SMA) which inserts into the lipids bilayer and forms small discs of bilayer (approx. 10nm diameter), with the polymer wrapped around the outside, termed SMA lipid particle (SMALPs)1,2. Proteins extracted within SMALPs retain their lipid bilayer environment, are more stable and are amenable to many downstream applications. Since then, the use of SMALPs has taken off around the world, and they have been used for high resolution structural studies, developing new assays and gaining novel insights to function3. However, there are some limitations to the approach, and despite the successes the molecular mechanism by which membrane extraction is achieved has not been established, making the development of improved polymers challenging4. This project will investigate the molecular mechanism of SMALP formation and determine which features of the polymer are important, using kinetics assays, light scattering techniques and EM imaging with a range of defined polymers, various lipids and example proteins. The long-term aim would be to use the knowledge obtained to design new polymers that retain the benefits of SMA but overcome the current limitations.

References

¹Knowles TJ, Finka R, Smith C, Lin YP, Dafforn T, Overduin M. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J Am Chem Soc. 2009 Jun 10;131(22):7484-5. doi: 10.1021/ja810046q.

²Jamshad M, Grimard V, Idini I, Knowles TJ, Dowle MR, Schofield N, Sridhar P, Lin YP, Finka R, Wheatley M, Thomas OR, Palmer RE, Overduin M, Govaerts C, Ruysschaert JM, Edler KJ, Dafforn TR. Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins. Nano Res. 2015 Mar;8(3):774-789. doi: 10.1007/s12274-014-0560-6.

³Unger L, Ronco-Campaña A, Kitchen P, Bill RM, Rothnie AJ. Biological insights from SMA-extracted proteins. Biochem Soc Trans. 2021 Jun 30;49(3):1349-1359. doi: 10.1042/BST20201067.

⁴Hawkins OP, Jahromi CPT, Gulamhussein AA, Nestorow S, Bahra T, Shelton C, Owusu-Mensah QK, Mohiddin N, O'Rourke H, Ajmal M, Byrnes K, Khan M, Nahar NN, Lim A, Harris C, Healy H, Hasan SW, Ahmed A, Evans L, Vaitsopoulou A, Akram A, Williams C, Binding J, Thandi RK, Joby A, Guest A, Tariq MZ, Rasool F, Cavanagh L, Kang S, Asparuhov B, Jestin A, Dafforn TR, Simms J, Bill RM, Goddard AD, Rothnie AJ. Membrane protein extraction and purification using partially-esterified SMA polymers. Biochim Biophys Acta Biomembr. 2021 Dec 1;1863(12):183758. doi: 10.1016/j.bbamem.2021.183758.

Techniques

• Cell culture & protein expression (bacterial, insect cell, mammalian cell)
• Light scattering kinetics
• Fluorescence spectroscopy
• Protein purification (affinity chromatography, SEC)
• SDS-PAGE & Western blots
• SAXS
• Electron microscopy