The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of
rhodopsin are associated with the
retinal degenerative disease autosomal dominant
retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding
proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound
retinal like wild-type
rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized
metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced
transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced
lysine. Increased formation of non-active forms of
rhodopsin, like
metarhodopsin III, may have some influence in the molecular defect underlying
retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of
rhodopsin.