A photoreversible conformational change in 124 kDa Avena phytochrome.
Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.
Singh
B R
BR
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Chai
Y G
YG
Song
P S
PS
Lee
J
J
Robinson
G W
GW
D020106:Acrylamide; D000178:Acrylamides; D000838:Anions; D002414:Cations, Monovalent; D002586:Cesium; D002712:Chlorides; D002523:Edible Grain; D005453:Fluorescence; D005456:Fluorescent Dyes; D008027:Light; D010834:Phytochrome / Q000528:radiation effects*; D010940:Plant Proteins / Q000528:radiation effects*; D011193:Potassium Iodide; D011487:Protein Conformation / Q000528:radiation effects; D012639:Seeds / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000178:Acrylamides; D000838:Anions; D002414:Cations, Monovalent; D002712:Chlorides; D005456:Fluorescent Dyes; D010940:Plant Proteins; D010834:Phytochrome; D011193:Potassium Iodide; D002586:Cesium; D020106:Acrylamide; D014364:Tryptophan; C028019:cesium chloride