Characterisation of styrene-maleic acid-solubilised G protein-coupled receptors

Abstract

Detergents have historically been used to solubilise membrane proteins for structural studies and pharmacological research, however detergents can alter the lipid environment surrounding a membrane protein. The styrene-maleic acid (SMA) copolymer has been designed to solubilise membrane proteins from the cell membrane, with the lipid bilayer intact, thus forming styrene-maleic acid lipid particles (SMALP). This would retain the native conformation of the protein and therefore suitable for applications such as drug discovery. In this project, the adenosine 2A receptor (A2AR) and the calcitonin gene-related peptide receptor (CGRPR) were solubilised into SMALPs. Various techniques were used to characterise the SMA-solubilised receptors.Out of the SMA copolymers tested, SMA2000 was chosen as the copolymer to solubilise the GPCRs. The copolymer was also compared with the new diisobutylene-maleic acid (DIBMA) copolymer, which has better resistance to divalent cations than the SMA copolymer. Molecular techniques confirmed the expression of the GPCRs in membranes and after solubilisation into SMALPs. Radioligand binding assays demonstrated that the A2AR retained its binding capability when solubilised and purified. The binding assay showed that the A2AR was more stable in SMALPs than DIBMA lipid particles (DIBMALP). Various techniques were used to characterise the A2AR-SMALP, providing novel properties of GPCRs in SMALP. The x-ray radiolytic footprinting (XRF) was used to detect regions of the GPCR-SMALP which were exposed to hydroxyl modification. The transmembrane domain, and the intracellular surface of the SMA-solubilised A2AR were exposed to water, demonstrating the SMALPs can successfully be used in XRF. Fluorescence correlation spectroscopy (FCS) was implemented to characterise the pharmacology of a single ligand binding to a single receptor, where the pharmacological profile of the A2AR was successfully characterised when in a SMALP.SMALPs were also tested for their applicability in phage display in order to generate GPCR-specific nanobodies against receptors in their native conformation. The M13 phage used in this project were conjugated with a VHH nanobody. The A2AR-SMALP was immobilised onto ELISA plates for phage binding, where approximately 22% of the total phage were A2AR-SMALP specific, which was lower than the control Fab protein. Avi-tagged A2AR and CGRPR constructs were designed to improve the immobilisation of the SMALPs, to yield a higher enrichment of phage, specific to the GPCR of interest.Finally, the photoaffinity cross-linking assay was implemented in this project, which has potential implications in drug discovery as receptors can be locked into a particular conformation when cross-linked with a ligand. The technique can theoretically be applied to receptors in SMALPs. Residues of the extracellular loops 1 and 3 of the CGRPR were studied. The assay showed residues A199, N200 and N201 of the extracellular loop 1 forming crosslinks with the ligand, when substituted with azidophenylalanine. Overall, the project demonstrated techniques which are applicable to study SMA-solubilised receptors. Using the various techniques revealed novel properties of the GPCRs in SMALPs. SMALPs were also applied to the drug discovery technique, phage display, with limited success. Techniques were incorporated into this study to improve the applicability of SMALPs in phage display.