Chromophore incorporation, Pr to Pfr kinetics, and Pfr thermal reversion of recombinant N-terminal fragments of phytochrome A and B chromoproteins.
N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (PthetaB; native chromophore) and phycocyanobilin (PCB). The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showed only a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics. With all apoproteins, PthetaB was incorporated faster than PCB. The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr is rapidly converted into Pr. Thus, neither the C-terminal domain nor homodimer formation regulates this property. Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots. The Pr to Pfr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I700 of phyA65-PCB decayed monoexponentially and the PthetaB analogue biexponentially, whereas the phyB66 I700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of Pfr from Pr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.
Max-Planck-Institut für Strahlenchemie, P.O. Box 101365, D-45413 Mülheim an der Ruhr, Germany.
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Remberg
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Ruddat
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Braslavsky
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W
Gärtner
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Schaffner
D001059:Apoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018554:Avena; D004926:Escherichia coli / Q000235:genetics; D007536:Isomerism; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism*; D010782:Photolysis; D010786:Photoreceptor Cells; D010834:Phytochrome / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051942:Phytochrome A; D051939:Phytochrome B; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D011198:Solanum tuberosum; D013053:Spectrophotometry; D013696:Temperature; D014157:Transcription Factors
D001059:Apoproteins; D010446:Peptide Fragments; D051942:Phytochrome A; D011994:Recombinant Proteins; D014157:Transcription Factors; D010834:Phytochrome; D051939:Phytochrome B