Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr-to-Pfr photoconversion.
Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1-595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the Pr-->Pfr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced Pr-->Pfr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I700 decay and accelerated Pfr formation. P318 is also involved in the Pr-->Pfr conversion, the millisecond steps (formation of Pfr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the Pr-->Pfr conversion with kinetics similar to wild-type protein.
Remberg
A
A
Max-Planck-Institut für Strahlenchemie, Mülheim an der Ruhr, Germany.
Schmidt
P
P
Braslavsky
S E
SE
Gärtner
W
W
Schaffner
K
K
D019943:Amino Acid Substitution; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics*; D018554:Avena; D001665:Binding Sites; D003062:Codon / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010777:Photochemistry; D010834:Phytochrome / Q000737:chemistry / Q000235:genetics* / Q000528:radiation effects; D051942:Phytochrome A; D011487:Protein Conformation / Q000528:radiation effects*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects
D001059:Apoproteins; D003062:Codon; D051942:Phytochrome A; D011993:Recombinant Fusion Proteins; D010834:Phytochrome