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首页> 外文期刊>Biochemistry >Self-assembly of recombinant prion protein of 106 residues.
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Self-assembly of recombinant prion protein of 106 residues.

机译：具有106个残基的重组病毒蛋白的自组装。

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摘要

The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrP(C)) to a beta-sheet-rich isoform (PrP(Sc)). The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrP(Sc) isoform. In an attempt to facilitate the structural analysis of PrP(Sc), a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Delta23-88 and Delta141-176) was expressed and purified from Escherichia coli. PrP106 retains the ability to support PrP(Sc) formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform [Supattapone, S., et al. (1999) Cell 96, 869-878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability. In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled beta-oligomer was observed. After proteinase K digestion, the oligomers retain an intact core with unusually high beta-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation. In contrast to the PrP(Sc)106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.
机译：病毒疾病发病机理中的中心事件是病毒蛋白质（PrP）从α螺旋（PrP（C））到富含β-sheet的同工型（PrP（Sc））的深刻构象变化。构象转变机理的阐明由于在相对不溶的易于聚集的PrP（Sc）同工型上收集高分辨率生物物理数据的挑战而变得复杂。为了促进PrP（Sc）的结构分析，表达并纯化了具有两个缺失（Delta23-88和Delta141-176）的106个氨基酸的编辑嵌合鼠仓鼠PrP（MHM2 PrP106）。 PrP106保留了在转基因小鼠中支持PrP（Sc）形成的能力，这意味着它包含所有PrP区域，这些区域是构象转变为致病同工型所必需的所有PrP区域[Supattapone，S.，等。 （1999）Cell 96，869-878]。重组未糖基化的PrP106（rPrP106）在低浓度下无结构，经历了浓度依赖性的构象转变，形成富含β-折叠的形式。构象转变后，rPrP106具有与PrP（Sc）106相似的性质，例如高β-折叠含量，确定的三级结构，对蛋白酶K有限的消化的抵抗力和高热力学稳定性。在GdnHCl诱导的变性研究中，观察到非结构化单体和组装的β-低聚物之间的单个协同构象过渡。蛋白酶K消化后，寡聚体保留了完整的核心，具有异常高的β-折叠含量（> 80％）。使用质谱法，我们发现rPrP106的134-215位残基区域受到蛋白酶K的保护，并具有不依赖溶剂的倾向，可以采用富含β-折叠的构象。与从神经功能障碍动物的大脑中纯化的PrP（Sc）106相反，多聚体β-rPrP106保持可溶，为详细的结构研究提供了机会。

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