Functional Expression and Site-Directed Mutagenesis of Photoactive Yellow Protein

The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1997-05, Vol.121 (5), p.876-880
Main Authors: Mihara, Ken'ichi, Hisatomi, Osamu, Imamoto, Yasushi, Kataoka, Mikio, Tokunaga, Fumio
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins - biosynthesis
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Binding Sites - genetics
Chromatiaceae - chemistry
chromophore/protein interaction
Electrophoresis, Polyacrylamide Gel
expression
in vitro mutagenesis
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed - genetics
opsin shift
photoactive yellow protein
Photoreceptors, Microbial
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Solubility
Spectrophotometry
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Summary:The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.
ISSN:0021-924X
DOI:10.1093/oxfordjournals.jbchem.a021668