Zhang H, Stockel J, Mehlhorn I, Groth D, Baldwin M A, Prusiner S B, James T L, Cohen F E
Department of Neurology, University of California, San Francisco 94143, USA.
Biochemistry. 1997 Mar 25;36(12):3543-53. doi: 10.1021/bi961965r.
PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.
已知PrP(Sc)是传染性朊病毒的主要成分，即便不是唯一成分。对PrP(Sc)进行有限蛋白酶解会产生一种N端截短的约142个残基的多肽，称为PrP 27-30。最近，一种对应于叙利亚仓鼠PrP 27-30的142个残基的重组蛋白（rPrP）在大肠杆菌中表达并纯化（Mehlhorn等人，1996年）。rPrP已在水性缓冲液中重折叠成α-螺旋和β-折叠结构以及各种中间体。通过圆二色光谱（CD）和核磁共振（NMR）对β-折叠状态和两种pH依赖性α-螺旋状态进行了表征。α-螺旋构象仅在分子内二硫键形成后出现，而β-折叠形式在有或无二硫键的情况下均可形成。在所研究的不同α-螺旋形式中，只有那些在pH值5-8范围内重折叠的在生理pH值下基本可溶，在上述整个pH范围内表现出相似的构象和单体分析沉降谱。此外，重折叠的α-rPrP显示出具有天然构象的蛋白质典型的NMR化学位移分散，尽管二维NMR表明存在大片段的构象灵活性。它表现出协同热变性转变；在升高的温度下，它迅速且不可逆地转变为热力学上更稳定的β-折叠形式。用盐酸胍（GdnHCl）使α-rPrP展开显示出两相转变，在2 M GdnHCl时有一个相对稳定的折叠中间体。第一相和第二相的ΔG值估计分别为1.9±0.4千卡/摩尔和6.5±1.2千卡/摩尔，这与被大量柔性构象片段包围的折叠核心一致。通过NMR，在pH 2下未重折叠分离得到的α-rPrP（酸性）表现出异质性线宽，这与酸变性的熔融球状状态一致。我们得出结论，就rPrP构成PrP(C)的相关折叠结构域而言，rPrP呈现的各种构象表明PrP序列在其构象上可能具有内在可塑性；实际上，PrP(C)的部分可能具有相对开放的构象，这使其在适当条件下易于转化为PrP(Sc)。
