Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and P_r‐to‐P_fr photoconversion

Abstract

Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1±595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the P r 3P fr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced P r 3P fr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I 700 decay and accelerated P fr formation. P318 is also involved in the P r 3P fr conversion, the millisecond steps (formation of P fr ) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the P r 3P fr conversion with kinetics similar to wild-type protein.Keywords: assembly; chromophore±protein interaction; heterologous expression; mutant; recombinant phytochrome.The phytochromes comprise a small protein family of ubiquitous photoreceptors in plants. They are activated upon light absorption by a covalently bound chromophore, the linear tetrapyrrole phytochromobilin (PFB) [1,2]. After the primary Z3E photoisomerization of the 15,16 double bond, a series of conformational changes of chromophore and protein convert the parent, physiologically inactive state (red-absorbing phytochrome, P r , l max = 667 nm) within milliseconds into the active far red-absorbing phytochrome (P fr ; l max = 730 nm) [3]. Both the photochemical reaction and the subsequent thermal processes of the chromophore are tuned by the protein environment. However, no three-dimensional structure of the protein is available which would allow exact identification of these chromophore±protein interactions. As alternative methods, variations in the chromophore structure [4±6] and site-directed mutagenesis (SDM) of the protein moiety have been employed. In view of the lack of information abouth the protein structure, SDM should preferably be confined to positions close to the chromophore-binding cysteine residue.[The position of the chromophore attachment in the presently best characterized phytochrome, phyA from oat (which is also dealt with here) is Cys321.] Mutagenesis has already revealed a strong influence of certain amino acid residues on the chromoprotein a...

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

“…Hence, characterization of the molecular interaction between the chromophore and the apoprotein is crucial for understanding the functional mechanism(s) of phytochromes. Several studies have addressed this interaction by using in vitro assembly of PHYA (5,28,29,47), PHYB (35,37,48), PHYC and PHYE (43), or apoprotein mutants (30)(31)(32). However, in contrast to the vertebrate photoreceptor rhodopsin (49), in the phytochrome field, little has been done to examine the relationships among chromophore structure, its assembly to apoprotein, and photochromism of the holoprotein, because of the difficulty of synthesizing the chromophore and its structural analogs.…”

“…Holophytochromes assembled with PCB showed photoreversible spectral change, although its peaks were slightly blue shifted when compared with the native phytochrome (27)(28)(29). Other strategies, including systematic N-and C-terminal truncations and site-directed mutagenesis of the apoprotein, have been used to study the structural requirements of the chromophore-apoprotein interaction in terms of photochromism (30)(31)(32).…”

Functional and Biochemical Analysis of the N-terminal Domain of Phytochrome A

“…Actually, the phyA-303 mutation also caused poor protein yield in the two heterologous systems tested here (data not shown). Poor protein yield in bacteria was also observed for the W366F mutated phyA (45).…”

“…Little is known, however, about the specific role played by different residues in the dual function of this domain as substrate and catalytic regulator. Slow or poor in vitro chromophore incorporation has been reported for proteins mutated close to the cysteine 323, between amino acids 309 and 326 (43)(44)(45). Based on the recently published crystal structure of the photosensory core of DrBphP (23), the latter region involves residues in close contact with the chromophore.…”

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

“…Some mutants displayed faster decays and accelerated P fr formation, whereas a proline to alanine mutation resulted in a slower P fr formation due to a more rigid conformation in the mutant. The acceleration was assigned to an increase in polarity or a weakened interaction between two protein domains enabling faster conformational changes (39). In SynCph2(1-2), the S385A mutation accelerated the formation of the red light-adapted state, where the decrease of polarity inhibits interactions that stabilize the intermediates.…”