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 ligh t 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 s tructural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this s tudy, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in qu antities suitable for spectroscopic and photochemical characterization. Whe n expressed in Drosophila, Rh1-ID4 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 p igment 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 kinet ics 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 fo rmed consisting of bathorhodopsin (lambda (max) = 545 nm) and isorhodopsin (lambda (max) = 462 nm). We also compared the spectroscopic and photochemic al properties of this pigment with other vertebrate pigments. We conclude t hat the binding site of Drosophila rhodopsin is similar to that of bovine r hodopsin and is characterized by a protonated Schiff base chromophore stabi lized via a single negatively charged counterion.