title: Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin.
abstract: Bacteriorhodopsin contains 8 tryptophan residues distributed across the membrane-embedded helices. To study their possible functions, we have replaced them one at a time by phenylalanine; in addition, Trp-137 and -138 have been replaced by cysteine. The mutants were prepared by cassette mutagenesis of the synthetic bacterio-opsin gene, expression and purification of the mutant apoproteins, renaturation, and chromophore regeneration. The replacement of Trp-10, Trp-12 (helix A), Trp-80 (helix C), and Trp-138 (helix E) by phenylalanine and of Trp-137 and Trp-138 by cysteine did not significantly alter the absorption spectra or affect their proton pumping. However, substitution of the remaining tryptophans by phenylalanine had the following effects. 1) Substitution of Trp-86 (helix C) and Trp-137 gave chromophores blue-shifted by 20 nm and resulted in reduced proton pumping to about 30%. 2) As also reported previously (Hackett, N. R., Stern, L. J., Chao, B. H., Kronis, K. A., and Khorana, H. G. (1987) J. Biol. Chem. 262, 9277-9284), substitution of Trp-182 and Trp-189 (helix F) caused large blue shifts (70 and 40 nm, respectively) in the chromophore and affected proton pumping. 3) The substitution of Trp-86 and Trp-182 by phenylalanine conferred acid instability on these mutants. The spectral shifts indicate that Trp-86, Trp-182, Trp-189, and possibly Trp-137 interact with retinal. It is proposed that these tryptophans, probably along with Tyr-57 (helix B) and Tyr-185 (helix F), form a retinal binding pocket. We discuss the role of tryptophan residues that are conserved in bacteriorhodopsin, halorhodopsin, and the related family of opsin proteins.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: T
authors.initials: T
authors.lastname: Mogi
authors.forename: T
authors.initials: T
authors.lastname: Marti
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008027:Light; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D011522:Protons; D012172:Retinaldehyde / Q000502:physiology*; D012176:Retinoids / Q000502:physiology*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000235:genetics / Q000502:physiology*
chemical_list: D011522:Protons; D012176:Retinoids; D010649:Phenylalanine; D001436:Bacteriorhodopsins; D014364:Tryptophan; D012172:Retinaldehyde