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A.F. X. Goldberg, L.M. Ritter, T. Arakawa; Predicted and measured disorder in the C–terminal domain of peripherin/rds. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2435.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Elaboration of vertebrate photoreceptor outer segments (OSs) requires peripherin/rds, a tetrameric integral membrane protein that results in a surprising variety of progressive retinal degenerations when defective. This protein acts as an essential structural element for OS architecture in a manner that is not currently understood. In contrast to findings for the structurally important EC2 extracellular domain, a high degree of intrinsic disorder is predicted for the peripherin/rds C–terminus, both by secondary structure and PONDR VL–XT analyses; this observation suggests that this region may possess an underlying conformational flexibility. Methods: We have characterized the protein’s hydrophilic C–terminal domain by expressing and purifying a recombinant fusion protein from E. coli. Milligram quantities of the 63 C–terminal amino acids of bovine peripherin/rds could be liberated and affinity–purified from a GST header by proteolysis with a site–specific protease. Results: At all protein concentrations examined, this domain (CTER) behaved as a monomer in aqueous solution, as determined by analytical ultracentrifugation AUC. Despite its high expression levels and solubility, a variety of approaches, including: intrinsic tryptophan fluorescence, size–exclusion chromatography, and far ultraviolet circular dichroism, indicate that CTER lacks significant tertiary structure and possesses limited secondary structure. Conclusions: Conformational flexibility predicted for the peripherin/rds C–terminus has been documented experimentally for a recombinant protein produced in E. coli. Current studies are directed at determining whether intrinsic disorder plays a role in situ for peripherin/rds structure and function.
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