Genetic basis of spectral tuning in the violet-sensitive visual pigment of African clawed frog, Xenopus laevis

Abstract

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (lambdamax) at approximately 360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV-VII on the lambdamax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.

Figure 1.

Schematic of the ancestral amphibian UV pigment, where TM helices are represented by rectangles with broken lines (Palczewski et al. 2000). NdeI, HindIII, BsmBI, SphI, and MfeI indicate the positions of the restriction enzyme recognition sites. The open and solid circles indicate the residues of identical and different amino acids between the ancestral and frog pigments. The shaded circles show the N and C termini of the chameleon UV pigment (for more details, see Shi and Yokoyama 2003).

Figure 2.

Absorption spectra of ancestral UV pigment (A), frog pigment (L), and their chimeric pigments (B–K). The pigment structures are shown in the top right. The residues of the ancestral pigment are shown as solid circles, those of the frog pigment as open circles, and those of chameleon N and C termini as shaded circles. The λ_max-values of the pigments are found in the middle. Note that the absorption spectra of a(67)f(99)a pigment (C) and f(99)a pigment (E) exhibit minor peaks at ∼420 nm.

Figure 3.

Schematic of the point mutations of the ancestral UV pigment. Solid circles indicate unchanged amino acid, whereas open circles indicate amino acid changes from the ancestral pigment to the frog pigment. The λ_max-values of the mutants are shown in the last column. The standard errors associated with these estimates are all within 1 nm. The λ_max-values with an asterisk (*) indicate that they have a second minor peak at ∼420 nm.

Figure 4.

Absorption spectra of the ancestral UV pigment with the seven mutations (A) and the frog violet pigment with the reverse mutations (B). The λ_max-values of the pigments are found in the middle right and the dark-light spectra in the inset.