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B. Nickle, S. Wilkie, J.A. Cowing, D.M. Hunt, P. Robinson; Variation In Constitutive Activation Among The Vertebrate Opsin Classes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):810.
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© ARVO (1962-2015); The Authors (2016-present)
Opsins, the protein component of photopigments, are classified into five classes for vertebrates. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS) and rod–opsin–like 1 and 2 (RH1, RH2). In rod–opsin two critical amino acids are K296, which binds the retinal based chromophore through a Schiff base, and E113, which acts as the counterion to the protonated Schiff base. Corresponding residues in each of the other four vertebrate opsin classes play similar roles. Using bovine rhodopsin, it has been shown that these two sites form a salt–bridge in the apoprotein which maintains the opsin in an inactive state. In RH1 opsins, disruption of this critical salt–bridge through mutation has been shown to shift the opsin into an active conformation that has the ability to constitutively activate transducin. We are interested in the conservation of this property across the other classes of vertebrate opsins.
Site directed mutagenesis was performed using QuickchangeTM. Mutant opsins are expressed in COS–1 cells, followed by isolation of the COS–1 membranes. GTPγS binding assays are carried out to assess activation of bovine transducin.
We demonstrate that mutations in site 113 and 296 produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their degree of constitutive activation.
The differences in constitutive activation profiles suggest different structural constraints exist among the vertebrate opsin classes. These constraints may lend to different activation mechanisms around the chromophore binding pocket can may provide further insight into rod and cone opsin differences.
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