Chromophore Incorporation, P_r to P_fr Kinetics, and P_fr 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 (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.

Both Subunits of the Dimeric Plant Photoreceptor Phytochrome Require Chromophore for Stability of the Far-red Light-absorbing Form

“…The former alternative is favored by our observation that phytochrome was occasionally completely stable for several hours even in living yeast cells (data not shown). Dark reversion of phytochrome is controlled by intramolecular properties; additionally, it appears to be modulated by external factors in plants (11,13). Our data demonstrate that some of these factors are also present in yeast and are not exclusively specific to plants.…”

“…The role of intra-subunit interactions varies. The difference spectra of monomeric, N-terminal fragments are blue-shifted for pea phyA but are unaltered for oat phyA and potato phyB (11,25).…”

“…The extent and rate of dark reversion vary widely between species and different phytochromes (8,9). Especially, phyA of monocotyledonous plants appears not to undergo dark reversion at all (10,11). In this case, like for other light labile phytochromes, signal attenuation is achieved by rapid degradation of the activated Pfr form (12).…”

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

“…Cph1 and CphA) are well characterized with respect to their photochemical activity (5,7,18,22,23). In contrast, photoreceptors such as SynCph2 (1)(2), composed of only two GAF domains in a tandem array and lacking the N-terminal PAS domain, have not been investigated before by time-resolved spectroscopy.…”

“…The excitation/ detection setup as employed here yields a step function in the wavelength range of the first intermediate (around 700 nm), as "lumi-R" or I 700 is formed within picoseconds (1) and remains constant far into the microsecond time range. The decay of this intermediate is described for group I phytochromes with a lifetime of ϳ80 -100 s (7,23). In group I phytochromes, the following intermediates (meta-R or I bleach ) show reduced oscillator strength (I bleach ), and the decay of the first intermediate follows monoexponential kinetics.…”