Molecular properties of chimerical mutants of gecko blue and bovine rhodopsin.
In spite of the high similarity in amino acid sequence between rod visual pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only the spectral sensitivities but also the thermal decay rates of the meta II- and III-intermediates are noticeably different from one another [Kojima et al. (1995) Biochemistry 34, 1096-1106]. In order to identify the protein region(s) that contain(s) key residue being responsible for the functional difference, we constructed six chimerical mutants derived from gecko blue and bovine rhodopsin, with the aid of protein production in a human embryonic kidney cell line (293S). While the absorption maximum of every mutant was located in between gecko blue (466 nm) and bovine rhodopsin (500 nm), a large blue-shift (18 nm) was observed when the helices I-III of rhodopsin were replaced with those of gecko blue. A time resolved spectroscopic study demonstrated that this replacement also accelerated the decay rate of the meta II-intermediate. The decay of the meta III-intermediate of the mutants became faster as the compartment of gecko blue was increased. Thus, the faster decay of the meta II-intermediate of gecko blue is largely attributed to residues within helices I-III, while the decay of the meta III-intermediate apparently depends on the overall structure of the protein.
Kojima
D
D
Department of Biophysics, Kyoto University, Sakyo-ku, Kyoto Japan.
Oura
T
T
Hisatomi
O
O
Tokunaga
F
F
Fukada
Y
Y
Yoshizawa
T
T
Shichida
Y
Y
D000818:Animals; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004563:Electrochemistry; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010777:Photochemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D012168:Retinal Pigments / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D017299:Rod Opsins; D013053:Spectrophotometry; D013816:Thermodynamics
D018076:DNA, Complementary; D005136:Eye Proteins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; C095853:short-wavelength opsin; D012243:Rhodopsin