Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris.
The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris. Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively). Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore. The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min. Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved. The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms.
Jorissen
H J M M
HJ
Max-Planck-Institut für Strahlenchemie, Mülheim an der Ruhr, Germany.
Braslavsky
S E
SE
Wagner
G
G
Gärtner
W
W
D001483:Base Sequence; D000460:Chlorophyta / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D010834:Phytochrome / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis
D017931:DNA Primers; D011994:Recombinant Proteins; D010834:Phytochrome