Binding of RPE65 fragments to lipid monolayers and identification of its partners by glutathione S-transferase pull-down assays

Biochemistry
Eric TrudelChristian Salesse

Abstract

RPE65 is the major component of the retinal pigment epithelium (RPE) microsomal membrane, and it plays a critical role in the binding of retinoids involved in the visual cycle. To understand how RPE65 binds to membranes, we have expressed and purified soluble fragments of human RPE65 fused to glutathione S-transferase (GST). The interaction between two fragments of RPE65 (F1 and F2 which include residues 1-125 and 126-250, respectively) and lipid monolayers has been studied by surface pressure, ellipsometry, and surface rheology measurements. Surface pressure and ellipsometry clearly showed a rapid adsorption of F2 to lipid monolayers whereas the kinetics of binding of F1 was much slower. Furthermore, the data suggest that the F2 fragment inserts into the lipid monolayer. Surface rheology showed a clear increase in monolayer rigidity only in the presence of F2, thereby demonstrating high intermolecular interactions of this fragment. This observation is further supported by the GST pull-down assays which demonstrated that F2 cosediments with full-length RPE65, suggesting that RPE65 has the propensity to form clusters or oligomers. The structure homology modeling of RPE65 based on a related family member, apocarotene 15',15'-oxyg...Continue Reading

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Citations

Oct 31, 2014·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Mustapha Lhor, Christian Salesse
Apr 23, 2011·Journal of Molecular Biology·Lionel ChièzeVéronique Vié
Mar 23, 2010·Progress in Retinal and Eye Research·Philip D Kiser, Krzysztof Palczewski
Sep 11, 2008·Journal of Theoretical Biology·Ralf Schmid
May 19, 2007·Colloids and Surfaces. B, Biointerfaces·Julie BoucherChristian Salesse
Dec 26, 2006·Archives of Biochemistry and Biophysics·Prakash Bhosale, Paul S Bernstein
Oct 1, 2016·Journal of the American Chemical Society·Philippe CalvezChristian Salesse
Apr 7, 2015·Biochemistry·Éric DemersChristian Salesse

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