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1.
Acta Crystallogr F Struct Biol Commun ; 80(Pt 3): 59-66, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38376821

RESUMO

Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1-Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1-655, 114-655 and 114-458, respectively), each C-terminally tagged with His6, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Šresolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Šresolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.


Assuntos
Fitocromo , Sorghum , Fitocromo B/genética , Sorghum/genética , Sorghum/metabolismo , Cristalização , Cristalografia por Raios X , Fitocromo/química , Fitocromo/genética , Fitocromo/metabolismo , Luz
2.
Nat Plants ; 6(5): 581-588, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32366982

RESUMO

Plant phytochromes are red/far-red photochromic photoreceptors that act as master regulators of development, controlling the expression of thousands of genes. Here, we describe the crystal structures of four plant phytochrome sensory modules, three at about 2 Å resolution or better, including the first of an A-type phytochrome. Together with extensive spectral data, these structures provide detailed insight into the structure and function of plant phytochromes. In the Pr state, the substitution of phycocyanobilin and phytochromobilin cofactors has no structural effect, nor does the amino-terminal extension play a significant functional role. Our data suggest that the chromophore propionates and especially the phytochrome-specific domain tongue act differently in plant and prokaryotic phytochromes. We find that the photoproduct in period-ARNT-single-minded (PAS)-cGMP-specific phosphodiesterase-adenylyl cyclase-FhlA (GAF) bidomains might represent a novel intermediate between MetaRc and Pfr. We also discuss the possible role of a likely nuclear localization signal specific to and conserved in the phytochrome A lineage.


Assuntos
Fitocromo/metabolismo , Plantas/metabolismo , Cristalografia por Raios X , Fitocromo/fisiologia , Estrutura Terciária de Proteína , Transdução de Sinais , Sorghum/metabolismo , Glycine max/metabolismo , Relação Estrutura-Atividade
3.
FEBS Lett ; 591(9): 1258-1265, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28376244

RESUMO

The N-terminal extension (NTE) of plant phytochromes has been suggested to play a functional role in signaling photoinduced structural changes. Here, we use resonance Raman spectroscopy to study the effect of the NTE on the chromophore structure of B-type phytochromes from two evolutionarily distant plants. NTE deletion seems to have no effect on the chromophore in the inactive Pr state, but alters the torsion of the C-D ring methine bridge and the surrounding hydrogen bonding network in the physiologically active Pfr state. These changes are accompanied by a shift of the conformational equilibrium between two Pfr substates, which might affect the thermal isomerization rate of the C-D double bond and, thus, account for the effect of the NTE on the dark reversion kinetics.


Assuntos
Fitocromo B/química , Fitocromo B/metabolismo , Plantas/metabolismo , Domínios Proteicos , Arabidopsis/genética , Arabidopsis/metabolismo , Sítios de Ligação/genética , Ligação de Hidrogênio , Cinética , Luz , Modelos Moleculares , Mutação , Fitocromo B/genética , Plantas/genética , Ligação Proteica/efeitos da radiação , Sorghum/genética , Sorghum/metabolismo , Análise Espectral Raman , Termodinâmica
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