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A.D. Albert, M. DeLivron, T. Stevens; Critical Phospholipids and Opsin Regeneration . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1738.
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Purpose: Solubilized opsin generally cannot be regenerated. In the detergent octylglucoside (OG) this loss of regenerability occurs when opsin is associated with less than 30 phospholipids. Furthermore, the regenerability exhibits a time dependent decay . CD studies suggested that α–helical secondary structure is lost upon bleaching solubilized rhodopsin . These observations suggest that solubilized rhodopsin is irreversibly denatured upon bleaching. In the present studies we address the following question. Can solubilized opsin be regenerated if it is allowed to reassociate with lipids? That is, is the denaturation of opsin reversible? Methods: Disk membranes were solubilized in OG. The solutions consisted of mixed OG micelles containing defined ratios of rhodopsin and phospholipid. The OG concentration was increased until rhodopsin was completely delipidated. The concentrations of OG required have been determined in great detail . The samples were bleached and an aliquot of the opsin regenerated by addition of 9–cis or 11–cis retinal. The samples were then dialyzed to remove the detergent and allow rhodopsin–lipid reassociation. After dialysis the opsin regenerability was again assessed. CD spectra were obtained also as a function of the phospholipid to rhodopsin ratio to monitor changes in secondary structure. Results: Consistent with earlier studies, regenerability was maintained until the phospholipids per rhodopsin (PL/R mole ratio) drops from approximately 70 to 30. At these PL/R ratios there is no evidence of change in the secondary structure. Between 30 and 15 PL/R the opsin is regenerable only if the phospholipids are allowed to reassociate with rhodopsin. However, opsin irreversibly looses its ability to regenerate if the PL/R ratio drops below 15. These results were mirrored by transitions in the CD.Conclusions:Not all membrane lipids are equal. A subset of approximately 30 phospholipids (enough to surround opsin) is required to support regeneration. If a subset of at least 15 phospholipids is associated with opsin, regenerability can be restored. That is, denaturation is reversible. However, loss of regenerability is irreversible if less than 15 phospholipids remain associated with opsin. These data suggest critical lipid–protein interaction for function and stability of rhodopsin. Interestingly, earlier studies showed that approximately 15 phospholipids per rhodopsin are in slow exchange with the bulk phospholipid in disk membranes .
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