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AD Albert, AF X Goldberg, M Kataragada, K Boesze-Battaglia; Interaction with the Lipid Surface Induces a Conformational Change in the C-terminal Region of Peripherin/rds . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1398.
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© ARVO (1962-2015); The Authors (2016-present)
Abstract: : Purpose: Membrane fusion proteins undergo conformational changes upon interaction with a lipid surface that allow them to become fusion competent. The far UV CD spectrum was used to monitor changes in the secondary structure of the C-terminus of peripherin/rds in the presence and absence of lipid. These studies allow us to characterize the fusion competent state of this protein. Methods: The wt and C-terminal truncation GST fusion proteins were engineered with a thrombin cleavage site. The proteins were cleaved from the GST using thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The biological activity of the purified proteins was assessed by depolarization studies, membrane leakage and membrane fusion studies. CD spectra of the wt peptide in 50mM phosphate buffer (pH 7) was obtained between 260nm and 200nm. DPC (dodecylphosphatidylcholine)was added in a 100:1 ratio to the peptide and the CD spectra again determined. Results: The purified wt C-terminal protein resolved as a monomer under reducing conditions and was immunoreactive with anti-peripherin/rds antibody 2B6 (gift from Dr. R. Molday). The purified wt protein was found to depolarize model membranes and to promote aqueous contents mixing, while the C-terminal truncation mutant was inactive. The far UV CD spectra indicated alpha helical content in the wt peptide. The increase in intensity of the 208nm and 222nm bands upon addition of DPC indicated an increase in helical content of the peptide in the presence of DPC. Conclusion: The C-terminal wt peptide exhibits alpha helical structure in aqueous buffer. The alpha helical secondary structure increases upon interaction with a lipid surface. This behavior of the wt C-terminal peptide is consistent with other fusion peptides.
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