Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.

The photobleaching sequence of a short-wavelength visual pigment.

The photobleaching pathway of a short-wavelength cone opsin purified in delipidated form (lambda(max) = 425 nm) is reported. The batho intermediate of the violet cone opsin generated at 45 K has an absorption maximum at 450 nm. The batho intermediate thermally decays to the lumi intermediate (lambda(max) = 435 nm) at 200 K. The lumi intermediate decays to the meta I (lambda(max) = 420 nm) and meta II (lambda(max) = 388 nm) intermediates at 258 and 263 K, respectively. The meta II intermediate decays to free retinal and opsin at >270 K. At 45, 75, and 140 K, the photochemical excitation of the violet cone opsin at 425 nm generates the batho intermediate at high concentrations under moderate illumination. The batho intermediate spectra, generated via decomposing the photostationary state spectra at 45 and 140 K, are identical and have properties typical of batho intermediates of other visual pigments. Extended illumination of the violet cone opsin at 75 K, however, generates a red-shifted photostationary state (relative to both the dark and the batho intermediates) that has as absorption maximum at approximately 470 nm, and thermally reverts to form the normal batho intermediate when warmed to 140 K. We conclude that this red-shifted photostationary state is a metastable state, characterized by a higher-energy protein conformation that allows relaxation of the all-trans chromophore into a more planar conformation. FTIR spectroscopy of violet cone opsin indicates conclusively that the chromophore is protonated. A similar transformation of the rhodopsin binding site generates a model for the VCOP binding site that predicts roughly 75% of the observed blue shift of the violet cone pigment relative to rhodopsin. MNDO-PSDCI calculations indicate that secondary interactions involving the binding site residues are as important as the first-order chromophore protein interactions in mediating the wavelength maximum.

Characterization of the primary photointermediates of Drosophila rhodopsin.

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.

Photochemistry of the primary event in short-wavelength visual opsins at low temperature.

Two short-wavelength cone opsins, frog (Xenopus laevis) violet and mouse UV, were expressed in mammalian COS1 cells, purified in delipidated form, and studied using cryogenic UV-vis spectrophotometry. At room temperature, the X. laevis violet opsin has an absorption maximum at 426 nm when generated with 11-cis-retinal and an absorption maximum of 415 nm when generated with 9-cis-retinal. The frog short-wavelength opsin has two different batho intermediates, one stable at 30 K (lambda(max) approximately 446 nm) and the other at 70 K (lambda(max) approximately 475 nm). Chloride ions do not affect the absorption maximum of the violet opsin. At room temperature, mouse UV opsin has an absorption maximum of 357 nm, while at 70 K, the pigment exhibits a bathochromic shift to 403 nm with distinct vibronic structure and a strong secondary vibronic band at 380 nm. We have observed linear relationships when analyzing the energy difference between the initial and bathochromic intermediates and the normalized difference spectra of the batho-shifted intermediates of rod and cone opsins. We conclude that the binding sites of these pigments change from red to green to violet via systematic shifts in the position of the primary counterion relative to the protonated Schiff base. The mouse UV cone opsin does not fit this trend, and we conclude that wavelength selection in this pigment must operate via a different molecular mechanism. We discuss the possibility that the mouse UV chromophore is initially unprotonated.