Constraints of the 9-Methyl Group Binding Pocket of the Rhodopsin Chromophore Probed by 9-Halogeno Substitution

Abstract

Sterical constraints of the 9-methyl-binding pocket of the rhodopsin chromophore are probed using retinal analogues carrying substituents of increasing size at the 9 position (H, F, Cl, Br, CH(3), and I). The corresponding 11-Z retinals were employed to investigate formation of photosensitive pigment, and the primary photoproduct was identified by Fourier transform infrared difference spectroscopy. In addition, any effects of cumulative strain were studied by introduction of the 9-Z configuration and/or the alpha-retinal ring structure. Our results show that the 9-F analogue still can escape from the 9-methyl-binding pocket and that its photochemistry behaves very similar to the 9-demethyl analogue. The 9-Cl and 9-Br analogues behave very similar to the native 9-methyl pigments, but the 9-I retinal does not fit very well and shows poor pigment formation. This puts an upper limit on the radial dimension of the 9-methyl pocket at 0.45-0.50 nm. Introduction of the alpha-retinal ring constraint in the 11-Z series results in cumulative strain, because the 9-I and 9-Br derivatives cannot bind to generate a photopigment. The 9-Z configuration can partially compensate for the additional alpha-retinal strain. The corresponding 9-Br analogue does form a photopigment, and the other derivatives give increased photopigment yields compared to the corresponding 11-Z derivatives. In fact, 9-Z-alpha-retinal would be an interesting candidate for retinal supplementation studies. Our data provide direct support for the concept that the 9-methyl group is an important determinant in ligand anchoring and activation of the protein and in general agree with a three-point interaction model involving the ring, 9-methyl group, and aldehyde function.