Influence of Expression System on Chromophore Binding and Preservation of Spectral Properties in Recombinant Phytochrome A

Abstract

N-Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C-terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation. The start and end positions of the cDNA employed (phyA from oat) mimic the positions of tryptic cleavage (deletion of the first 64 amino acids, and stop codons after amino acid positions 425 or 595, generating 39-kDa and 59-kDa peptides, respectively). The absorption properties and photochromicity upon redfar-red irradiation of these mutants were compared with their tryptic counterparts derived from native oat phytochrome and with recombinant products possessing intact N-termini, but C-terminal positions identical to those of the corresponding tryptic fragments (45-kDa and 65-kDa peptides). All recombinant 65-kDa and 59-kDa peptides bound the chromophore after expression and showed the appropriate absorption spectra of the P, and the P,, forms. The smaller chromopeptides (45-kDa and 39-kDa) behaved differently depending on the expression system employed. E. coli-derived peptides exhibited a phytochrome-like difference spectrum only when the intact N-terminus was present (45-kDa product). The recombinant 39-kDa peptide from E. coli was incapable of chromophore binding whereas the identical peptide sequence expressed by fl pastoris formed a chromoprotein with phycocyanobilin. This recombinant phytochrome fragment exhibited a difference spectrum (Pr-P,) with an even larger P, absorption band than the comparable tryptic 39-kDa fragment. Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the PJP,, absorption characteristics. Evidently, trypsin digestion down to the 39-kDa fragment affects protein conformation also in terms of P, conservation.

Studies on the Structure and Function of Phytochromes as Photoreceptors Based on Synthetic Organic Chemistry

“…Several groups have reported in vitro assembly of PhyB and have shown that it has similar spectral properties to PhyA. [134][135][136] To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we designed and chemically synthesized more than 20 bilin chromophore derivatives (Fig. 10), including natural PÈB and PCB, and reconstituted them with recombinant Arabidopsis PhyB apoprotein (PHYB) through collaborative study with biologists.…”

“…Finally, the deprotection of the allyl ester moiety of 134 was achieved by Pd 0 -catalyzed reaction 47,49 using sodium p-toluenesulfinate as a nucleophile in THF/MeOH to give the desired locked chromophore 122 (15Za-18Et-BV) in 90% yield. 54,115 2.10.3 Synthesis of the E-syn CD-Ring Component and 15Es-18Et-BV: 5-Tosylpyrrolinone derivative 114 was prepared starting from propenal (135) as shown in Scheme 32. Treatment of 135 with acetic acid in the presence of a catalytic amount of zinc acetate afforded 3-acetoxypropanal (136) in 40% yield.…”

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

“…In contrast, PhyB has not been well characterized because of its low abundance in native tissue. Recently, several groups reported in vitro assembly of PhyB and showed that it has similar spectral properties to PhyA (35)(36)(37).…”

Light modulation of vegetative development.

“…The image shows the thioether linkage that connects the chromophore to the Cys at amino acid position -320 (322 of oat region was sufficient for photosensory function (Lagarias and Rapoport, 1980;Boylan et al, 1994). Transgenic plant and recombinant expression experiments have refined these original conclusions and established that the 40-kD region flanking Cys-322 is sufficient for chromophore attachment and photoreversibility (Cherry et al, 1993;Gartner et al, 1996). This work also showed that there is a blue shift of the Pfr absorption maximum and increased nonphotochemical reversion of Pfr to Pr when a 6-to 10-kD region at the N terminus is removed, suggesting that this region may be involved in establishing the proper chromophore-protein environment (Cherry et al, 1992).…”

“…Two types of studies have been performed on the photochemically active N-termina1 chromophore binding domain-overexpression in transgenic plants, and in vitro assembly of truncated or mutant recombinant phytochromes in yeast (Deforce et ai., 1991 ;Wahleithner et al, 1991 ;Cherry et al, 1993;Kunkel et al, 1993;Hill et al, 1994;Gartner et al, 1996). Earlier studies had established that Cys-322 of oat PHYA was responsible for chromophore binding and that the 70-kD N-terminal (A) The two spectrally photointerconvertible forms of phytochrome, Pr and Pfr.…”