Phytochrome assembly. Defining chromophore structural requirements for covalent attachment and photoreversibility.
Assembly of holophytochrome in the plant cell requires covalent attachment of the linear tetrapyrrole chromophore precursor, phytochromobilin, to a unique cysteine in the nascent apoprotein. In this investigation we compare chromophore analogs with the natural chromophore precursor for their ability to attach covalently to recombinant oat apophytochrome and to form photoactive holoproteins. Ethylidene-containing analogs readily form covalent adducts with apophytochrome, whereas chromophores lacking this double bond are poor substrates for attachment. Kinetic measurements establish that although the chromophore binding site on apophytochrome is best tailored to phytochromobilin, apophytochrome will accommodate the two analogs with modified D-rings, phycocyanobilin and phycoerythrobilin. The phycocyanobilin-apophytochrome adduct is photoactive and undergoes a light-induced protein conformational change similar to the native holoprotein. By contrast, the phycoerythrobilin adduct is locked into a photochemically inactive protein conformation that is similar to the red light-absorbing Pr form of phytochrome. These results support the hypothesis that the photoconversion from Pr to Pfr, the far red light- absorbing form of phytochrome, involves the photoisomerization of the C15 double bond. Knowledge gained from these studies provides impetus for rational design of chromophore analogs whose insertion into apophytochrome should elicit profound changes in light-mediated plant growth and development.
Li
L
L
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Lagarias
J C
JC
D001059:Apoproteins / Q000737:chemistry; D001654:Bile Pigments / Q000737:chemistry; D002523:Edible Grain; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010834:Phytochrome / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins; D013057:Spectrum Analysis
D001059:Apoproteins; D001654:Bile Pigments; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010834:Phytochrome