Studies on Plant Bile Pigments, VII. Preparation and Characterization of Phycobiliproteins with Chromophores Chemically Modified by Reduction

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.

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“…pastoris expression system was obtained from Invitrogen. Phycocyanobilin was purified as described (Kufer and Scheer, 1979).…”

Section: Methodsmentioning

confidence: 99%

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

Gärtner

Hill

Worm

et al. 1996

European Journal of Biochemistry

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“…The two maxima of B-PE at 545 and 565 nm, for example, are shifted to one single, broad maximum, at 555nm, while the absorbance is only slightly diminished. This is comparable to the denaturation effect of low concentrations of urea (Langer er a/., 1980;Kufer and Scheer, 1979). The recovery at low temperatures would then be due to an increased rigidity ('resolidification') of the chromophores.…”

Section: Mechanismmentioning

confidence: 79%

“…C-Phycoerythrin was isolated from Spirulina platensis (Kufer and Scheer, 1979), B-phycoerythrin (B-PE) (KostReyes and Kost, 1979) and phycobilisomes (PBS) from Porphyridiurn cruentum (Gantt and Lipschultz, 1972). Stock solutions of the lyophilized biliproteins were prepared in tris buffer (0.01 M, pH 7.7) and diluted immediately before measurements.…”

Section: Methodsmentioning

confidence: 99%

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Phycobilisomes and Isolated Phycobiliproteins. Effect of Glutardialdehyde and Benzoquinone on Fluorescence

Köst¹,

Rath²,

Wanner³

et al. 1981

Photochem & Photobiology

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Abstract-The fluorescence of the biliproteins C-phycocyanin from Spirulina platensis, B-phycoerythrin from Porphyridium cruentum and of isolated whole P. cruentum phycobilisomes is quenched in the presence of glutardialdehyde (GA) or benzoquinone (BQ). The kinetics of fluorescence decrease thus induced is biphasic. If GA is used as a quencher, the fluorescence can be recovered at 77 K. Contrary to the GA-effect, only a minor recovery takes place with BQ at 77K, thus demonstrating a different mechanism of action of GA and BQ on biliproteins.

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“…Chromophore Preparation-Phycocyanobilin (PCB) was extracted from Spirulina platensis as described (32). Biliverdin IX␣ was derived from bilirubin by oxidation (33) and was purified by high pressure liquid chromatography with the column Inertsil-3-C 18 Plasmid Construction and Plant Transformation-Construction of the PHYA-65-WT-GFP-GUS-NLS chimeric gene was performed in several steps fusing all the four genes.…”

Section: Methodsmentioning

confidence: 99%

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

Mateos

Luppi

Ogorodnikova

et al. 2006

Journal of Biological Chemistry

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Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 Å ) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, ␤-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.Plant photoreceptors monitor the cues provided by the light environment and trigger modifications that adjust growth and development to the prevailing conditions (1, 2). Phytochromes are sensors of red and far-red (FR) 3 light that bind an open chain tetrapyrrole chromophore (3, 4). Arabidopsis thaliana bears five phytochrome apoprotein genes (PHYA through PHYE) (5). phyA is a versatile photoreceptor that induces seed germination in response to brief exposures to light (6, 7), which the seeds may experience during soil labor. phyA is also required to perceive the prolonged exposures to FR that the seedlings experience when they emerge from the soil under dense plant canopies (8). These two photoresponses mediated by phyA are of the types called very low fluence responses (VLFR) and high irradiance responses (HIR), respectively (9). Some physiological processes (e.g. inhibition of hypocotyl growth, unfolding of the cotyledons) exhibit both VLFR and HIR as two discrete phases of response, where VLFR saturates with infrequent FR pulses and HIR requires very frequent or continuous FR (10). HIR requires cis-acting elements at target gene promoters (11) and domains of the phyA molecule itself (12) that are dispensable for VLFR.The N-terminal domain of phytochromes bears the chromophore attachment site (13) and provides differential spectral selectivity to phyA (more active under FR than red light) compared with phyB (more active under red light than FR) (14). The C-terminal domain contains a histidine kinase-related sequence motif that is able to mediate phosphorylation (15, 16), two PAS (domain named after Per, Arnt, and Sim) motifs important for downstream signaling in the context of the full molecule (17), and residues necessary for dimerization (18,19) and targeting to the nucleus upon light activation (20, 21)...

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Studies on Plant Bile Pigments, VII. Preparation and Characterization of Phycobiliproteins with Chromophores Chemically Modified by Reduction

Cited by 72 publications

References 38 publications

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

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

Phycobilisomes and Isolated Phycobiliproteins. Effect of Glutardialdehyde and Benzoquinone on Fluorescence

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

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