Two Amyloid States of the Prion Protein Display Significantly Different Folding Patterns
Two Amyloid States of the Prion Protein Display Significantly Different Folding Patterns
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DOI：
10.1016/j.jmb.2010.05.051
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发表时间：
2010-07-23
影响因子：
5.6
通讯作者：
Baskakov, Ilia V.
中科院分区：
文献类型：
--
作者：
Ostapchenko, Valeriy G.;Sawaya, Michael R.;Baskakov, Ilia V.
已经确定的是，单个氨基酸序列可以产生几种构象上不同的淀粉样蛋白状态。然而，在同一序列中形成的淀粉样结构的不同程度仍然不清楚。为了解决这个问题，我们研究了两种淀粉样蛋白状态（称为R-和S-原纤维）在体外产生的高度纯化的全长重组朊病毒蛋白。几种生物物理技术，包括X-射线衍射，CD，傅里叶变换红外光谱（FTIR），氢氘交换，蛋白酶K消化，并结合构象敏感的荧光染料显示，R-和S-原纤维有显着不同的二级，三级和四级结构。这两个国家都显示了4。8-通过典型的淀粉样蛋白交叉β-棘的埃角X射线衍射，它们显示出明显不同的赤道轮廓，表明β-链的不同折叠模式。通过FTIR监测的氢-氘交换实验表明，只有一小部分酰胺质子在R-或S-原纤维中受到保护，这是其交叉β-结构的动态性质的一个论据。尽管如此，这两种淀粉样蛋白状态被发现是非常稳定的构象，从温度诱导的变性FTIR和构象敏感染料监测判断。当加热到80摄氏度时，只有局部的展开被揭示出来，而个别状态特异性的交叉β特征被保留下来。目前的研究表明，由相同的氨基酸序列形成的两种淀粉样蛋白状态表现出显着不同的折叠模式，这可能反映了两种不同的交叉β结构的架构。然而，这两种沙R-原纤维，共享高构象稳定性，认为蛋白质折叠和聚集的能量景观可以包含几个深自由能极小值。(C)2010爱思唯尔有限公司版权所有。
It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question, we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein. Several biophysical techniques including X-ray diffraction, CD, Fourier transform infrared spectroscopy (FTIR), hydrogen-deuterium exchange, proteinase K digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary, and quaternary structures. While both states displayed a 4. 8-angstrom meridional X-ray diffraction typical for amyloid cross-beta-spines, they showed markedly different equatorial profiles, suggesting different folding pattern of beta-strands. The experiments on hydrogen-deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-beta-structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 degrees C, only local unfolding was revealed, while individual state-specific cross-beta features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-beta-structure. Both Sand R-fibrils, however, shared high conformational stability, arguing that the energy landscape for protein folding and aggregation can contain several deep free-energy minima. (C) 2010 Elsevier Ltd. All rights reserved.