Mutagenesis studies of human red opsin: Trp-281 is essential for proper folding and protein-retinal interactions

Human red and green opsins contain a strikingly large number of tryptophan residues. These tryptophans are highly conserved among all red and green op sins. To investigate possible roles of these tryptophans in folding and str ucture, we have systematically replaced each tryptophan of human red opsin. When expressed in COS cells, wild-type red opsin undergoes N-linked glycos ylation, forms a light-sensitive pigment with absorption maximum at 560 nm upon reconstitution with Il-cis-retinal, and is transported to the plasma m embrane. We used the extent of glycosylation, pigment generation, and intra cellular localization of mutant red opsins as our criteria for assessing th e effect of substitution. Replacement of eight tryptophans, Trp-59, Trp-90, Trp-149, Trp-152, Trp-183, Trp-191, Trp-195, and Trp-243, with Phe or Ala did not affect the wild-type phenotype significantly. However, replacement of Trp-5 and Trp-51 in the putative N-terminal domain and Trp-142, Trp-177, Trp-179, and Trp-281 in the transmembrane domain with Phe had profound eff ects, indicating that these substitutions affected red opsin folding, Judge d by the severity of the effects, we propose that Trp-5, Trp-51, Trp-177, a nd Trp-281 are important for red opsin folding. Although substitution of Tr p-281 with Phe and Cys did not permit normal glycosylation and transport, s ubstitution with Tyr and His permitted these processes but resulted in blue -shifted pigment. Thus, polar aromatics appear to substitute for Trp-281 to allow red opsin folding. The large spectral shift indicates that Trp-281 i s essential for the proper interaction of the protein with 11-cis-retinal.