| Popis: |
Detergent-free solubilisation using a polymer of styrene maleic acid (SMA) has proven useful in the study of membrane proteins. SMA was employed to solubilise G-protein-coupled receptors (GPCRs) from mammalian cells, into SMA lipid particles (SMALPs). Optimal SMALP solubilisation conditions were determined to be 2 % (w/v) SMA, at 37 °C for 1 h retaining wildtype (WT)-like pharmacological profiles and conferring improved stability on GPCRs over detergent micelles. Endoplasmic reticulum (ER)-retention motifs –KDEL and –KHILFRRRRRGFRQ were found not to be applicable to all proteins. Study of SMALP-solubilisation of intracellular membranes was prevented by the inability to retain the adenosine 2a receptor (A2aR) in the ER. A cysteine-nul A2aR construct was produced containing two reporter groups and behaved as WT-A2aR. This construct is ready for use in fluorescence studies to further understanding of A2aR and the use of SMALPs in biophysical techniques. A range of GPCRs, from different GPCR subfamilies, were SMALP-solubilised with retention of ligand binding capability. Methods were successfully developed to reduce non-specific binding arising from ionic interactions of ligand and SMA. Finally, in a world first, the SMA analogue styrene maleimide, was shown to solubilise GPCRs with retention of ligand binding capability. |
