Abstract
Purpose: :
RPE65 is a key enzyme of the visual cycle, converting all-trans retinyl ester to 11-cis retinol. Association of RPE65 with the membrane causes a change of the RPE65 conformation and is required for its isomerohydrolase activity.The purpose of this study is to evaluate the effect of the different phospholipids on the association of the RPE65 with the phospholipid membrane.
Methods: :
Recombinant chicken RPE65 with a His-tag was expressed in 293 cells stably expressing LRAT using an adenovirus vector and purified to homogeneity. The RPE65 isomerohydrolase activity was quantified by HPLC using retinyl palmitate incorporated into liposomes. Fluorescent spectroscopy was used to evaluate an effect of the phospholipids nature on the depth of insertion of the RPE65 into the membrane bilayer. Liposomes composed of either DOPS (50 mol%) or DOPC (50 mol%) and containing 35 mol% of PSPC with the internal fluorescence quenchers were used to test the insertion depth.
Results: :
The quenching of RPE65 tryptophan fluorescence emission was evaluated after RPE65’s association with the liposomes containing the internal quenchers situated at different depths within the liposome’s bilayer. In the presence of DOPC liposomes, the RPE65 tryptophan emission was quenched by the quencher attached to the 6,7-positions of the phospholipid fatty acid acyl chain, whereas no quenching was observed from the quenchers located at either the 9,10- or 11,12-positions. However, upon the association with DOPS liposomes, the RPE65 tryptophan emission quenching was observed for the quenchers located at the 6,7-, 9,10- and 11,12-positions. The quenching efficiency was higher in the presence of the DOPS liposome containing the quencher at 6,7-positions in comparison to that with the quencher at 9,10- or 11,12-positions.
Conclusions: :
The results of the RPE65 tryptophan emission changes demonstrated that the depth of the RPE65’s insertion into the phospholipids membrane bilayer depends on a phospholipids type. These results reveal the novel effect of phospholipids nature on the RPE65 - membrane association and, in turn, contribute to the understanding of the RPE65 enzymatic mechanism.
Keywords: protein structure/function • lipids • retinoids/retinoid binding proteins