Site-directed incorporation of p-nitrophenylalanine into streptavidin and site-to-site photoinduced electron transfer from a pyrenyl group to a nitrophenyl group on the protein framework
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作者:
Murakami, H
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Okayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, JapanOkayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, Japan
Murakami, H
[1
]
Hohsaka, T
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Okayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, JapanOkayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, Japan
Hohsaka, T
[1
]
Ashizuka, Y
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Okayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, JapanOkayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, Japan
Ashizuka, Y
[1
]
Sisido, M
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Okayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, JapanOkayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, Japan
Sisido, M
[1
]
机构:
[1] Okayama Univ, Fac Engn, Dept Biosci & Biotechnol, Okayama 7008530, Japan
Site-directed mutagenesis of streptavidin was carried out using a frame-shift suppressor tRNA that is aminoacylated with L-p-nitrophenylalanine and has a CCCG 4-base anticodon. Streptavidins carrying a single p-nitrophenylalanine at 22 different sites were prepared from mRNAs that contain a single CGGG 4-base codon at the mutation site. Of the 22 mutants, 14 mutants were found to bind N-biotinyl-L-l-pyrenylalanine. Site-to-site photoinduced electron transfer from the excited pyrenyl group to the nitrophenyl group was observed with steady-state fluorescence spectroscopy as well as by fluorescence decay measurement. The rate constants of the electron transfer decreased with the edge-to-edge distances that were predicted from the X-ray crystallographic structure of streptavidin. The distance dependence was analyzed on the basis of the tunneling pathway model. It was found that the rate constants are compatible with those in other proteins carrying metal complexes, except for the cases where electron transfer occurs through long space.