双色高粱植物色素 B (phyB) 光感模块的表达、纯化和结晶。

IF 1.1 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Acta crystallographica. Section F, Structural biology communications Pub Date : 2024-03-01 Epub Date: 2024-02-20 DOI:10.1107/S2053230X24000827
Sintayehu Manaye Shenkutie, Soshichiro Nagano, Jon Hughes
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引用次数: 0

摘要

高粱是一种短日照热带植物,已适应温带谷物生产,特别是通过在成熟度基因座(Ma1-Ma6)上选择变体,降低了对光周期的敏感性。Ma3 编码植物色素 B(phyB),它是一种红/远红光色素双蛋白光感受器。该多域基因产物由 1178 个氨基酸组成,能自动催化与植物色素的发色团结合,形成具有光活性的全植物色素(Sb.phyB)。本研究描述了一种高效异源过量生产系统的开发过程,该系统可大量生产各种整体蛋白构建体,以及纯化和结晶过程。我们成功制备出了 NPGP、PGP 和 PG 的 Pr(吸收红光)形式晶体(残基分别为 1-655、114-655 和 114-458),每种晶体都在 C 端用 His6 标记。NPGP 晶体没有衍射,而 PGP 和 PG 晶体的衍射分辨率分别为 6 Å 和 2.1 Å。将标签移至 N 端,并用藻蓝蛋白取代藻蓝蛋白作为配体,制备出的 PG 晶体衍射分辨率为 1.8 Å。这些结果表明,通过移除柔性区域、移动融合标签和改变小分子配体,可以提高具有挑战性的蛋白质晶体的衍射质量。
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Expression, purification and crystallization of the photosensory module of phytochrome B (phyB) from Sorghum bicolor.

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 Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Å resolution. 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.

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来源期刊
Acta crystallographica. Section F, Structural biology communications
Acta crystallographica. Section F, Structural biology communications BIOCHEMICAL RESEARCH METHODSBIOCHEMISTRY &-BIOCHEMISTRY & MOLECULAR BIOLOGY
CiteScore
1.90
自引率
0.00%
发文量
95
期刊介绍: Acta Crystallographica Section F is a rapid structural biology communications journal. Articles on any aspect of structural biology, including structures determined using high-throughput methods or from iterative studies such as those used in the pharmaceutical industry, are welcomed by the journal. The journal offers the option of open access, and all communications benefit from unlimited free use of colour illustrations and no page charges. Authors are encouraged to submit multimedia content for publication with their articles. Acta Cryst. F has a dedicated online tool called publBio that is designed to make the preparation and submission of articles easier for authors.
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