title: Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin.
abstract: Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Kim
authors.forename: P J
authors.initials: PJ
authors.lastname: Booth
authors.forename: S J
authors.initials: SJ
authors.lastname: Allen
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002793:Cholic Acids / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D019811:Hydroxylamine / Q000378:metabolism; D017136:Ion Transport / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010742:Phospholipid Ethers / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D002793:Cholic Acids; D008823:Micelles; D010742:Phospholipid Ethers; D011522:Protons; D011994:Recombinant Proteins; D019811:Hydroxylamine; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D012172:Retinaldehyde; D004134:Dimyristoylphosphatidylcholine