Abstract
Purpose: :
RPE65 is a key enzyme in the vertebrate's visual cycle, accomplishing hydrolysis and isomerization of all-trans retinyl ester to 11-cis retinol. To catalyze the reaction, purified recombinant RPE65 is required to bind to a phospholipid membrane. The purpose of this study is to investigate the specificity of RPE65's interaction with the phospholipid membrane.
Methods: :
Recombinant RPE65 carrying a 6×His tag was expressed in HEK293A-LRAT cells using an adenovirus vector and purified utilizing Ni-NTA affinity chromatography. Circular dichroism (CD) and a liposome flotation assay were used to assess the conformation of RPE65 and the specifisity of RPE65's interaction with a phospholipid membrane employing both the plain and the all-trans retinyl palmitate-containing liposomes. The dependence of the enzymatic activity of purified RPE65 on the phospholipid composition of the liposomes was analyzed by HPLC.
Results: :
Investigation of the RPE65's secondary structure using CD spectroscopy showed that RPE65 displayed a spectrum characteristic of -helix and β-sheet structures with considerable amounts of random-coil conformations. Secondary structure predictions indicated an approximate 40% of -helical, 15% of β-sheet and 45% random-coil conformation in purified RPE65 in the absence of liposomes. However, the addition of phospholipid unilamellar vesicles (liposomes) induced an alteration in the secondary structure of RPE65, showing a change in -helical and β-sheet content dependent on the nature of the phospholipids. Using a liposomes flotation assay, it was found that the RPE65 binding affinity towards liposomes was related on the phospholipid composition of liposomes. Moreover, RPE65 binding to the DOPC vesicles was significantly enhanced when all-trans retinyl palmitate was incorporated into the liposomes. The impact of the phospholipid nature on isomerohydrolase activity of RPE65 was studied using liposomes containing various phospholipids. The 11-cis retinol formation was not apparently dependent on liposomes composition.
Conclusions: :
RPE65 changes its conformation upon binding with the phospholipids membrane. The RPE65 binding affinity to the membrane depends on the liposomes composition and seems to be enhanced by the all-trans retinyl ester incorporation.
Keywords: protein structure/function • retinoids/retinoid binding proteins • lipids