| Rapid Retinal Release from a Cone Visual Pigment Following Photoactivation. | |
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MedLine Citation:
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PMID: 22217337 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Abstract. As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated retinal release from a short-wavelength sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t1/2) of retinal release from VCOP was 7.1 s, 250-fold faster than rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t1/2 decreasing from 23 s to 4 s with pH 4.1 to 8, respectively. However, the Arrhenius activation energy (Ea) for VCOP derived from kinetic measurements between 4<sup>o</sup> and 20<sup>o</sup>C was 17.4 kcal/mol, similar to 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D<sub>2</sub>O) effect in VCOP, but less than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP<sup>D108A</sup>) produced a pigment with an unprotonated chromophore (λmax = 360 nm) and dramatically slowed (t1/2 ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D/ D108A) was designed to move the counterion one alpha helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λmax = 420 nm). Moreover, VCOP<sup>S85D/D108A</sup> mutant had retinal release kinetics (t1/2 = 7 s) and Ea (18 kcal/mol) similar to the native pigment exhibiting no pH-dependence. By contrast, the single mutant VCOP<sup>S85D</sup> mutant had a ~3-fold decrease in retinal release rate compared to the native pigment. Photoactivated VCOP<sup>D108A</sup> had kinetics comparable to a rhodopsin counterion mutant, Rho<sup>E113Q</sup>, both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis, but rather from differences in the non-covalent binding properties of the retinal chromophore to the protein. |
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Authors:
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Min-Hsuan Chen; Colleen Kuemmel; Robert Richards Birge; Barry E Knox |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-1-3 |
Journal Detail:
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Title: Biochemistry Volume: - ISSN: 1520-4995 ISO Abbreviation: - Publication Date: 2012 Jan |
Date Detail:
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Created Date: 2012-1-5 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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