Urea unfolding of opsin in phospholipid bicelles.
Opsin is the unstable apo-protein of the light-activated G protein-coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/detergent bicelles, against urea-induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far-UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in alpha-helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2-dihexanoyl-sn-glycero-3-phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin.
McKibbin
Craig
C
Department of Biochemistry, University of Bristol, Bristol, UK. craig.mckibbin@manchester.ac.uk
Farmer
Nicola A
NA
Edwards
Patricia C
PC
Villa
Claudio
C
Booth
Paula J
PJ
D000818:Animals; D002417:Cattle; D002793:Cholic Acids; D004134:Dimyristoylphosphatidylcholine; D007700:Kinetics; D055355:Opsins / Q000737:chemistry* / Q000378:metabolism*; D010742:Phospholipid Ethers; D010743:Phospholipids / Q000737:chemistry*; D017510:Protein Folding; D013053:Spectrophotometry; D014364:Tryptophan; D014508:Urea
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D002793:Cholic Acids; D055355:Opsins; D010742:Phospholipid Ethers; D010743:Phospholipids; D014364:Tryptophan; D014508:Urea; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004134:Dimyristoylphosphatidylcholine