title: Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity.
abstract: The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.
authors: Department of Physiology, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA.
authors: Olga
authors: O
authors: Nikolaeva
authors: Gennadiy
authors: G
authors: Moiseyev
authors: Karla K
authors: KK
authors: Rodgers
authors: Jian-Xing
authors: JX
authors: Ma
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens; D004224:Diterpenes; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D000084562:Retinyl Esters; D014801:Vitamin A / Q000031:analogs & derivatives / Q000378:metabolism; D019745:cis-trans-Isomerases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004224:Diterpenes; D005136:Eye Proteins; D008081:Liposomes; D008563:Membrane Lipids; D000084562:Retinyl Esters; C022521:retinol-palmitate esterase; D014801:Vitamin A; C014794:retinol palmitate; D002265:Carboxylic Ester Hydrolases; D019745:cis-trans-Isomerases