Photoreversible change in the conformation of phytochrome as probed with a covalently bound fluorescent sulfhydryl reagent, N-(9-acridinyl)maleimide

Abstract

A fluorescent SH reagent, N-(9-acridiny1)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 (P r) and the far-red-light absorbing form (P fr). 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- rwas greater than that of NAM-P fr, indicating that the Cys residues modified by NAM were in more hydrophobic environment in P fr than in P r. The FI of the conjugate prepared from a red-light-irradiated sample of phytochrome was greater than that for the conjugate prepared from P r, indicating that more Cys residues were modifiable in P fr than P r. 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 photoconversio n of P r to P fr. However, it did not change upon subsequent photoconversion between P r and P fr. These results suggest that the initial increase in FI may have resulted from the binding of additional ANS to P fr and that the microenvironment of bound ANS may not be influenced by the photoconversion of phytochrome.