Dedicated to Professor Albert Eschenmoser on the occasion of his 75th birthday
The retinal chromophore (11Z)-3-diazo-4-oxoretinal (1) with two photo-labile moieties has been synthesized by semi-hydrogenation of an 11-yne precursor with activated Zn in aqueous media. Incorporation of 1 into opsin yielded diazoketo rhodopsin (DK-Rh), which, upon bleaching, gave rise to intermediates batho-Rh, lumi-Rh, meta-Rh, and meta-II-Rh corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates showed that the ionone ring cross-linked to Trp265 of helix F in DK-Rh and batho intermediate, and to Ala169 of helix D in lumi, meta-I, and meta-II intermediates. These results demonstrate the occurrence of large conformational changes along the visual transduction path, which, in turn, is responsible for activation of the G-protein.
Introduction. ± Rhodopsin (Rh) is a seven-transmembrane a-helical G-Protein-Coupled Receptor (GPCR) composed of 348 amino acids and the chromophore, (11Z)-retinal, which is bound to Lys296 as a protonated Schiff base [1] [2]. It is found in the outer segment of vertebrate rods, and is the photoreceptor that mediates dim vision. Visual transduction is triggered by photons which isomerize the (11Z)-chromophore to (all-E); this isomerization triggers a chain of conformational changes in the opsin, which induces an enzymatic cascade leading to vision [2 ± 4]. Scheme 1 depicts the intermediates present in the visual transduction process of Rh, identified by flash photolysis and various low-temperature spectroscopic measurements [4] [5]. Light irradiation of Rh results in (11Z) 3 (11E) isomerization of the chromophore. This yields photo-Rh (femtosecond process), which is converted into batho-Rh, a primary product that can be sequestered at À 1408; in contrast to Rh, l max 500 nm, batho-Rh absorbs at 543 nm and is considered to adopt a highly-strained (11E)-double bond [1] [3]. As shown in Scheme 1, batho-Rh relaxes thermally to further photo intermediates, i.e., lumi-Rh, that is in equilibrium with the blue-shifted intermediate, and then to meta-I-Rh and meta-II-Rh.
We have been interested in tracing the path of photo-isomerization and to identify the relative position of the chromophore with respect to Rh at each photointermediate. Results to date have shown that the C(3) of the ionone ring is in close contact with helix F of Rh in the dark prior to photo isomerization [4] [5]. However, the contacts made by the chromophore after light activation, which subsequently lead to visual transduction, remain to be clarified. Recent site-directed spin labeling of rhodopsin mutants (and disulfide cross-linking) [6] have demonstrated that movements