Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin
- Massachusetts Institute of Technology, Cambridge (USA)
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their possible functions, we have replaced them one at a time by phenylalanine; in addition, Trp-137 and -138 have been replaced by cysteine.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, 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.
- OSTI ID:
- 5608135
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 264:24; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
RHODOPSIN
STRUCTURE-ACTIVITY RELATIONSHIPS
AMINO ACID SEQUENCE
CYSTEINE
MEMBRANE PROTEINS
MUTATIONS
PHENYLALANINE
PROTEIN STRUCTURE
PROTONS
RETINOIC ACID
TRYPTOPHAN
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BARYONS
CARBOXYLIC ACID ESTERS
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ESTERS
FERMIONS
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INDOLES
MOLECULAR STRUCTURE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PIGMENTS
PROTEINS
PYRROLES
THIOLS
550200* - Biochemistry