Chromophore Incorporation, Pr to Pfr Kinetics, and Pfr Thermal Reversion of Recombinant N-Terminal Fragments of Phytochrome A and B Chromoproteins

Abstract

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 (PΦB; 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, PΦB was incorporated faster than PCB. The thermal stabilities of the P_fr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 P_fr is highly stable, whereas potato phyB66 P_fr is rapidly converted into P_r. 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 P_r to P_fr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I₇₀₀ of phyA65-PCB decayed monoexponentially and the PΦB analogue biexponentially, whereas the phyB66 I₇₀₀ decayed monoexponentially irrespective of the chromophore incorporated. The formation of P_fr from P_r 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.