Abstract by Ciara Frances Pugh

Amphiphilic copolymers enable the detergent-free extraction of membrane proteins from cellular membranes by encapsulating them within lipid nanodiscs, commonly referred to as native nanodiscs. This approach aims to preserve the native lipid environment and interaction partners of membrane proteins, thereby allowing biochemical and biophysical analyses under physiologically relevant conditions. The application of poly(styrene-co-maleic acid) (SMA) copolymers for characterising membrane proteins has spearheaded this field. However, SMA copolymers have inherent limitations, including a narrow working pH range, sensitivity to divalent cations, and high monomer dispersity. These drawbacks have driven the development of novel copolymers to improve upon the existing system. Despite these developments, few high-resolution structures of membrane proteins in native nanodiscs have been determined, with only a small subset originating from mammalian sources. This underscores the need to advance the field of amphiphilic copolymer research, with a particular focus on structural and functional studies of membrane proteins in native nanodiscs. 

In this thesis, a novel amphiphilic copolymer, poly(methacrylic acid-co-styrene) termed MAASTY, was developed. MAASTY copolymers were evaluated for their e!ectiveness as solubilising agents and their applicability for facilitating structural and functional studies of membrane proteins. With a focus on single particle cryogenic electron microscopy studies, these copolymers were used to stabilise eukaryotic membrane proteins, expressed in mammalian cells, in native nanodiscs, enabling high-resolution structural determination. Membrane proteins encapsulated by MAASTY copolymers retained activity, thus demonstrating compatibility with pharmacological characterisation. Additionally, the copolymers exhibited versatility in solubilising diverse membrane lipid compositions and a broad range of membrane proteins expressed across various cellular systems. MAASTY copolymers were further engineered through biotin conjugation to demonstrate their potential for functionalisation and immobilisation on solid supports. As part of continued development, the influence of copolymer sequence on solubilisation efficiency was systematically assessed, revealing opportunities for targeted optimisation of MAASTY copolymer design and for the rational design of novel next-generation copolymers. Finally, the use of amphiphilic copolymers as alternatives to detergents for biochemical assays was promoted through the utility of MAASTY copolymers for detecting membrane proteins via detergent-free enzyme-linked immunosorbent assays. Together, the findings of this thesis establish MAASTY copolymers as valuable additions to the existing library of published copolymers and demonstrate their significant potential for advancing membrane protein research.