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E. Trudel, S. Beaufils, A. Renault, R. Breton, C. Salesse; Characterization of the Membrane Interactions of Human RPE65 Fragments . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5138.
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Purpose: RPE65, the major component of the RPE microsomal membrane, plays a critical role in the binding of retinoids involved in the visual cycle. Since the discovery of this protein, many efforts have been deployed to express and purify RPE65 for structural and functional analysis. RPE65 is known to be closely associated with membranes. Consequently, to understand how RPE65 binds to membranes, we have expressed and purified soluble fragments of human RPE65 fused to glutathione–s–transferase (GST). Methods: Previous studies have shown that the full length RPE65 expressed in E.coli was insoluble. On the other hand, expression in insect cells produces a soluble form of RPE65 which is, however, very little soluble. Three human RPE65 fragments were thus cloned in fusion with GST and expressed in E.coli (fragments 1, 2 and 3 correspond to residues 1 to 125, 126 to 250, 251 to 533, respectively). After purification to homogeneity, fragments 1 and 2 show a high solubility without detergent. These two fragments were studied by ellipsometry and surface rheology using lipid monolayers as membrane models. Ellipsometry and surface rheology allow to respectively measure with high sensitivity the adsorption kinetics and the increase of the rigidity of these protein fragments. GST pull down assays were performed by binding RPE65 fragments to glutathione sepharose beads to cosediment proteins from RPE cell extracts which specifically bind to RPE65. Results: Surface pressure and ellipsometry clearly showed a rapid adsorption of fragment 2 to monolayers whereas the kinetics of binding of fragment 1 is much slower. Furthermore, data suggests that this fragment inserts into the lipid monolayer. Surface rheology shows a clear increase in rigidity only with fragment 2, demonstrating high intermolecular interactions. This observation is further supported by the GST pull–down assays demonstrating that fragment 2 cosediment with RPE65. Conclusions: This study shows that fragment 2 interacts much stronger with lipid monolayers than does fragment 1, which suggests that fragment 2 should be most important for membrane binding by RPE65. Moreover, given that these fragments are not acylated, these data also suggest that post–translational modifications are not essential for an effective binding of RPE65 to membranes. Furthermore, surface rheology and GST pull–down assays indicate that fragment 2 could be responsible for intermolecular cohesion such as clustering or oligomerization.
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