Chromophore Incorporation, Pr to Pfr Kinetics, and Pfr Thermal Reversion of Recombinant N-Terminal Fragments of Phytochrome A and B Chromoproteins
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- 作者：Anja Remberg ; Antje Ruddat ; Silvia E. Braslavsky ; Wolfgang Gä ; rtner ; and Kurt Schaffner
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：July 14, 1998
- 年：1998
- 卷：37
- 期：28
- 页码：9983 - 9990
- 全文大小：93K
- 年卷期：v.37,no.28(July 14, 1998)
- ISSN：1520-4995
文摘
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 yeastsSaccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (P
B; nativechromophore) and phycocyanobilin (PCB). The phyA65 apoprotein from yeast showed a monoexponentialassembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showedonly a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore moreslowly than the phyA65s, with biexponential kinetics. With all apoproteins, PB was incorporated fasterthan PCB. The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known forthe full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr israpidly converted into Pr. Thus, neither the C-terminal domain nor homodimer formation regulates thisproperty. 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 I700of phyA65-PCB decayed monoexponentially and the PB analogue biexponentially, whereas the phyB66I700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of Pfr fromPr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvementof the C-terminal domain in the relatively slow protein conformational changes.
B; nativechromophore) and phycocyanobilin (PCB). The phyA65 apoprotein from yeast showed a monoexponentialassembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showedonly a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore moreslowly than the phyA65s, with biexponential kinetics. With all apoproteins, PB was incorporated fasterthan PCB. The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known forthe full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr israpidly converted into Pr. Thus, neither the C-terminal domain nor homodimer formation regulates thisproperty. 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 I700of phyA65-PCB decayed monoexponentially and the PB analogue biexponentially, whereas the phyB66I700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of Pfr fromPr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvementof the C-terminal domain in the relatively slow protein conformational changes.