Spectral perturbations and oligomer/monomer formation in 124-kilodalton Avena phytochrome.
We have studied the effects of pH, ionic strength, and hydrophobic fluorescence probes, 8-anilinonaphthalene-1-sulfonate (ANS) and bis-ANS, on the structure of intact (124-kDa) Avena phytochrome. The Pfr form of phytochrome forms oligomers in solution to a greater extent than the Pr form. Hydrophobic forces play a major role in the oligomerization of phytochrome, as suggested by fluorescence and monomerization by bis-ANS. However, electrostatic charges also take part in the phytochrome oligomerization. The partial proteolytic digestion patterns for the Pr and Pfr species are different, but binding of bis-ANS to the phytochrome abolishes this difference and yields an identical proteolytic peptide mapping for both spectral forms of phytochrome. This appears to result from bis-ANS binding at the carboxy-terminal domain, which induces monomerization of phytochrome oligomers. A second bis-ANS binding at an amino-terminal site blocks cleavage sites of trypsin and alpha-chymotrypsin. Bis-ANS especially blocks access of the proteases to the amino-terminal cleavage site that produces an early proteolytic product (114/118 kDa) on SDS gels. The bis-ANS binding does not, however, affect the proteolytic cleavage site that occurs in the hinge region between the two structural domains of phytochrome, the chromophore domain and the C-terminal non-chromophore domain. A chromophore binding site in the Pfr form is apparently exposed for preferential binding of bis-ANS, causing cyclization of the chromophore and bleaching of its absorbance at 730 nm. These observations have been discussed in terms of a photoreversible topographic change of the chromophore/apoprotein during the phototransformation of phytochrome.
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
J K
JK
Choi
I S
IS
Kim
T I
TI
Kwon
W
W
Parker
P S
PS
Song
D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D002523:Edible Grain; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009994:Osmolar Concentration; D010447:Peptide Hydrolases; D010777:Photochemistry; D010834:Phytochrome / Q000528:radiation effects; D010940:Plant Proteins / Q000528:radiation effects; D011487:Protein Conformation; D012996:Solutions; D013053:Spectrophotometry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D010940:Plant Proteins; D012996:Solutions; D010834:Phytochrome; D010447:Peptide Hydrolases