Title : Mutagenesis studies of human red opsin:
trp-281 is essential for proper folding and protein-retinal interactions
Abstract :
- Human red and green opsins contain a strikingly large number of tryptophan residues
- These tryptophans are highly conserved among all red and green opsins.
- To investigate possible roles of these tryptophans in folding and structure, we have systematically replaced each tryptophan of human red opsin
- When expressed in COS cells, wild-type red opsin undergoes N-linked glycosylation, forms a light-sensitive pigment with absorption maximum at 560 nm upon reconstitution with 11- cis-retinal, and is transported to the plasma membrane
- We used the extent of glycosylation, pigment generation, and intracellular localization of mutant red opsins as our criteria for assessing the 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 effects, indicating that these substitutions affected red opsin folding
- Judged by the severity of the effects, we propose that Trp-5, Trp-51, Trp-177, and Trp-281 are important for red opsin folding
- Although substitution of Trp-281 with Phe and Cys did not permit normal glycosylation and transport, substitution 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 is essential for the proper interaction of the protein with 11- cis-retinal