Baskakov I V, Aagaard C, Mehlhorn I, Wille H, Groth D, Baldwin M A, Prusiner S B, Cohen F E
Institute for Neurodegenerative Diseases, Department of Neurology, University of California, San Francisco 94143, USA.
Biochemistry. 2000 Mar 14;39(10):2792-804. doi: 10.1021/bi9923353.
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.
朊病毒疾病发病机制的核心事件是朊病毒蛋白（PrP）从α-螺旋形式（PrP(C)）向富含β-折叠的异构体（PrP(Sc)）发生深刻的构象变化。由于难以收集关于相对不溶性且易于聚集的PrP(Sc)异构体的高分辨率生物物理数据，构象转变机制的阐明变得复杂。为了便于对PrP(Sc)进行结构分析，从大肠杆菌中表达并纯化了一种经过编辑的106个氨基酸的嵌合小鼠-仓鼠PrP（MHM2 PrP106），它有两个缺失区域（Δ23 - 88和Δ141 - 176）。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PrP中所有对于向致病异构体进行构象转变所必需的区域[Supattapone, S.,等人（1999年）《细胞》96卷，869 - 878页]。重组无糖基化的PrP106（rPrP106）在低浓度下无结构，会经历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相似的特性，如高β-折叠含量、确定的三级结构、对蛋白酶K有限消化的抗性以及高热力学稳定性。在盐酸胍诱导的变性研究中，观察到无结构单体与组装的β-寡聚体之间发生单一的协同构象转变。蛋白酶K消化后，寡聚体保留了一个完整的核心，其β-折叠含量异常高（>80%）。通过质谱分析，我们发现rPrP106的134 - 215位残基区域对蛋白酶K消化具有抗性，并且具有与溶剂无关的形成富含β-折叠构象的倾向。与从神经受损动物大脑中纯化的PrP(Sc)106不同，多聚体β-rPrP106保持可溶，为详细的结构研究提供了机会。
