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C. M. Kuemmel, M.-H. Chen*, B. E. Knox; Retinal Release of Cone Visual Pigment. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1664.
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© ARVO (1962-2015); The Authors (2016-present)
To gain an understanding of the mechanism of retinal release in cone visual pigments, we examined both bovine rhodopsin and Xenopus violet cone opsin (VCOP) proteins including primary counterion mutants.
VCOP and rhodopsin site specific mutations were generated and then expressed in COS-1 cells using transient transfections. Purified protein was tested for steady-state fluorescence measurements. An external light source was used for visual pigment photoactivation during data collection. Proteins were evaluated at pH 4.1, 6.0 and 8. Fluorescence measurements were performed between 4 and 20.4 oC.
The release of retinal is a necessary step in recovery from photoactivation for visual pigments. The rate of retinal release in VCOP was found to be ~250 fold faster than in rhodopsin. Yet VCOP and rhodopsin exhibited similar activation energy (Ea) for retinal release. In the absence of the primary counterion, the amount of retinal release was significantly reduced in VCOP. Reintroduction of a negative charge to an adjacent helix in the absence of the primary counterion restored the amount of retinal release and the Ea to similar levels as wild type (WT) VCOP. But, the reaction occurred at a slower rate. WT VCOP exhibited a strong pH dependence on the rate and the Ea for retinal release. The VCOP primary counterion mutant with a negative charge placed on an adjacent helix did not exhibit a pH dependent rate of retinal release. However pH did influence the Ea. Both VCOP and rhodopsin mutants lacking the primary counterion partially release retinal after a prolonged period following initial photoactivation, compared with respective WT proteins. Only after hydroxylamine addition was there complete release of retinal.
Similar Ea for VCOP and rhodopsin suggests the energetic barrier in release is the same for both visual pigments. Fluorescence measurements of visual pigment mutants support that the negative primary counterion is necessary for efficient release of retinal and Schiff base cleavage. Schiff base hydrolysis thus appears to be an important step in retinal release and may be the energetic barrier in the mechanism of retinal release by visual pigments. Interhelical residues within the VCOP retinal binding pocket are currently being investigated for a possible role in retinal release.
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