M. Han et al., ROLE OF THE C-9 METHYL-GROUP IN RHODOPSIN ACTIVATION - CHARACTERIZATION OF MUTANT OPSINS WITH THE ARTIFICIAL CHROMOPHORE 11-CIS-9-DEMETHYLRETINAL, Biochemistry, 37(2), 1998, pp. 538-545
Activation of the visual pigment rhodopsin involves both steric and el
ectrostatic interactions between the chromophore and opsin within the
retinal-binding site. Removal of the C-9 methyl group of 11-cis-retina
l inhibits light-dependent activation of the G protein, transducin, su
ggesting a direct steric contact. More recently, we have shown that st
eric interactions lead to receptor activation when Gly(121) in the mid
dle of transmembrane helix 3 is replaced by larger hydrophobic residue
s. In order to understand in more detail the role of the C-9 methyl gr
oup of retinal in the structure and function of rhodopsin, we first st
udied the properties of recombinant 9-dm-Rho (opsin reconstituted with
11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shif
ted lambda(max), increased hydroxylamine reactivity, and decreased abi
lity to activate transducin. These properties are consistent with the
hypothesis that the C-9 methyl group is a crucial structural anchor or
the correct docking of the chromophore in its binding site. Next, we
investigated the possible interaction between Gly(121) of opsin and th
e C-9 methyl group of retinal by characterizing recombinant pigments p
roduced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11
-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind
the chromophore. However, the double mutant G121L/F261A bound 11-cis-
9-demethylretinal to form a stable pigment with a lambda(max) of 451 n
m. When activity was assayed in membranes, the reduction in transducin
activation by 9-dm-Rho caused by the lack of a C-9 methyl group on th
e chromophore could be partially restored by replacing Gly(121) with a
bulky residue (leucine, isoleucine, or tryptophan). These results sup
port a model of receptor activation that involves steric interaction b
etween the C-9 methyl group of the chromophore and the opsin in the vi
cinity of Gly(121) on transmembrane helix 3.