Surface-Tethered Styrene Maleic Acid Lipid Particles (SMALPS)

: Towards the Application of Membrane Proteins in Devices

Abstract

Membrane proteins (MPs) are invaluable to the development of medicine and cellular biology, yet their tendency to fall apart outside of the cell has precluded implementation in sensors and other biotechnologies. SMA, a copolymer of styrene and maleic acid, is uniquely able to extract MPs directly from cells and in doing so preserves the weak stabilising interactions within the native membrane. This has allowed for the structure and function of a number of MPs to be better characterised in a more natural state, without the need for detergents or crystallisation. However, the wide uptake of often poorly-synthesised, free radical copolymer variants has meant a dearth of information remains over the exact mechanisms of interaction between the copolymer and lipids and how the polymeric microstructure may affect this. Increasingly, controlled RAFT polymerisation is used to synthesise SMA with well-defined molecular weights and comonomer sequences. Opportunistically, this method also imparts copolymers with chemically-unique end groups which express broad reaction chemistry for quantitative functionalisation. By exploiting this and other means, the research presented in this thesis investigated strategies by which novel functionality may be introduced to SMA, to both expand upon current applications and to better understand the workings of the copolymer in these systems. Functionalisation of the end group was also explored and interfering with the polarity of these groups was found to significantly affect the global properties of the copolymers as well as their propensity to form copolymer aggregates or lipid nanodiscs. The nanostructures formed by the copolymers were assessed using a suite of techniques including interfacial measurements, microscopy, light and small angle neutron scattering and the formation of nanodiscs evaluated by turbidimetric measurements using both model DMPC vesicle and E. coli membrane suspensions. Deeper appreciation of these behaviours was gained by the preparation of copolymers with equivalent molecular weights, but with inverted block sequences in respect to the end groups. This allowed surface functionalisation to be investigated, where the copolymer block sequence, perpendicular to either nanoparticle or solid substrate interfaces, could be controlled. SMA-functionalised magnetic iron oxide nanoparticles were achieved by grafting the copolymer from RAFT agents used as the stabilising ligand. This was able to separate lipid vesicles and gramicidin from mixed aqueous suspensions, useful as an uncomplicated extraction method. A graft-to approach was used to modify Au nanoparticles and substrates by selectively reducing the RAFT end group to a thiol. Capacitance measurements using functionalised Au electrodes could detect the adsorption of lipid layers and the activity of gramicidin channels, stabilised at the interface. Ultimately, the work aimed to demonstrate that RAFT-made SMA presents a potentially straightforward route to the fabrication of technologies using SMALPs as a generic support for native membranes, or membrane proteins.

Date of Award	24 Apr 2024
Original language	English
Awarding Institution	University of Bath
Supervisor	Pedro Estrela (Supervisor), Karen Edler (Supervisor), Gareth Price (Supervisor) & Paul Whitley (Supervisor)