title: Opsin stability and folding: modulation by phospholipid bicelles.
abstract: Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-alpha-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to approximately 70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.
authors.affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. craig.mckibbin@manchester.ac.uk
authors.forename: Craig
authors.initials: C
authors.lastname: McKibbin
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Farmer
authors.forename: Chris
authors.initials: C
authors.lastname: Jeans
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: H Gobind
authors.initials: HG
authors.lastname: Khorana
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Edwards
authors.forename: Claudio
authors.initials: C
authors.lastname: Villa
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D002942:Circular Dichroism; D008051:Lipid Bilayers; D010743:Phospholipids / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017299:Rod Opsins / Q000737:chemistry*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
chemical_list: D008051:Lipid Bilayers; D010743:Phospholipids; D017299:Rod Opsins