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 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.
Nakayama
T A
TA
Department of Biostructure and Function, University of Connecticut Health Center, Farmington 06030-3705, USA. tnakayam@neuron.uchc.edu
Zhang
W
W
Cowan
A
A
Kung
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D019556:COS Cells; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000737:chemistry / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; C095855:long-wavelength opsin; D010649:Phenylalanine; D014364:Tryptophan