Regulation of Phototransduction in Short-Wavelength Cone Visual Pigments via the Retinylidene Schiff Base Counterion

Abstract

Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.

Vertebrate ultraviolet visual pigments: Protonation of the retinylidene Schiff base and a counterion switch during photoactivation

“…Expression constructs and COS1 cell transfections were described previously (16). MUV samples had A 357 Ϸ 0.3-0.4 with an A 280 ͞A max ratio of Ϸ4, and the E108Q mutant had A 354 Ϸ 0.3-0.4 and an A 280 ͞A max ratio of Ϸ4.5.…”

“…Light-dependent activation of bovine transducin by using filter binding assays were carried out as described (16,18).…”

“…The Xenopus SWS1 cone pigment (VCOP, max Ϸ 427 nm) has a protonated SB in the dark and remains so until formation of the active species (14)(15)(16). Neutralizing the conserved acidic residue (D108) in TM3 converts the violet-sensitive pigment into a UV-sensitive pigment ( max Ϸ 353 nm), consistent with a drop in the pK a and deprotonation of the SB (16). The VCOP-D108A mutant does not activate transducin in the dark but has a significantly prolonged R* lifetime.…”

Genetic Basis of Spectral Tuning in the Violet-Sensitive Visual Pigment of African Clawed Frog, Xenopus laevis

“…When T93P, L116V, and S118T are introduced into the ancestral pigment, they do not cause any significant l maxshift individually (Figure 3). Babu et al (2001) have found that the reverse mutation, D113E, in the frog pigment decreases the l max -value by 12 nm. Much to our surprise, however, when E113D is introduced into the ancestral pigment, the mutant pigment also decreases the l max by 4 nm (Figure 3), a direction that is the opposite of that predicted from the previous observation.…”

“…The molecular analyses of avian and mammalian pigments show that the differentiations of UV and violet pigments have occurred by the accumulations of various amino acid replacements at a total of 11 sites, 46,49,52,86,90,93,97,113,114,116, and 118 in TM helices I-III, where amino acid site numbers are standardized by those of the bovine rhodopsin (Wilkie et al 2000;Yokoyama and Shi 2000;Babu et al 2001;Shi et al 2001;Cowing et al 2002;Fasick et al 2002;Shi and Yokoyama 2003;Parry et al 2004;Yokoyama and Tada 2005;Yokoyamaet al 2005b). Furthermore, engineered ancestral pigments of a wide range of vertebrates show that most vertebrate ancestors, including the common ancestor, had UV pigments.…”