Photoreversible change in the conformation of phytochrome as probed with a covalently bound fluorescent sulfhydryl reagent, N-(9-acridinyl)maleimide.
A fluorescent SH reagent, N-(9-acridinyl)maleimide (NAM), was used as a fluorescent probe to detect changes in the conformation of phytochrome from pea (Pisum sativum) seedlings during photoconversion between the red-light absorbing form (Pr) and the far-red-light absorbing form (Pfr). NAM-Cys conjugates emitted weaker fluorescence in non-polar solvent than in polar solvent. The fluorescence intensity (FI) of NAM-phytochrome conjugates depended on the absorbing form of phytochrome: the FI of NAM-Pr was greater than that of NAM-Pfr, indicating that the Cys residues modified by NAM were in more hydrophobic environment in Pfr than in Pr. The FI of the conjugate prepared from a red-light-irradiated sample of phytochrome was greater than that for the conjugate prepared from Pr, indicating that more Cys residues were modifiable in Pfr than Pr. The fluorescence polarization of the conjugate (0.0251) indicates that the modified Cys residues may be located at the surface of the phytochrome molecule. The FI of phytochrome conjugates with 8-anilinonaphthalene-1-sulfonate (ANS) did not change with the photoconversion of phytochrome. The FI of a mixture of ANS and phytochrome increased upon the first photoconversion of Pr to Pfr. However, it did not change upon subsequent photoconversion between Pr and Pfr. These results suggest that the initial increase in FI may have resulted from the binding of additional ANS to Pfr and that the microenvironment of bound ANS may not be influenced by the photoconversion of phytochrome.
Division of Biological Regulation, National Institute for Basic Biology, Okazaki, Japan.
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Yamamoto
D007887:Fabaceae / Q000378:metabolism; D005456:Fluorescent Dyes; D008027:Light; D008301:Maleimides; D015335:Molecular Probes; D010834:Phytochrome / Q000528:radiation effects*; D010946:Plants, Medicinal; D011487:Protein Conformation / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents
D005456:Fluorescent Dyes; D008301:Maleimides; D015335:Molecular Probes; D013439:Sulfhydryl Reagents; D010834:Phytochrome; C080490:N-(9-acridinyl)maleimide