Preparation and characterization of purified amyloid fibrils.
Preparation and characterization of purified amyloid fibrils.
复制标题
纯化淀粉样原纤维的制备和表征。
DOI：
10.1021/ja016229b
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发表时间：
2001
影响因子：
15
通讯作者：
C. Dobson
中科院分区：
文献类型：
--
作者：
J. Zurdo;J. Guijarro;C. Dobson
淀粉样原纤维是高度组织化的蛋白质聚集体，与阿尔茨海默病和海绵状脑病等致病性疾病有关。它们也被探索为具有广泛潜在应用的新型纳米结构。尽管在这类原纤维的表征方面取得了相当大的进展，但其结构的许多重要方面仍有待阐明。其中包括淀粉样蛋白纤维内蛋白质链的构象状态的详细定义以及组成原丝的组装方式。研究这些问题的研究受到体外制备的纤维样品的内在异质性的阻碍，4因为这些样品总是含有可溶性前体和非纤维聚集体。因此，开发分离原纤维的策略是非常重要的。在这里，我们报告了一种方法来实现这一目标，并用它来表征在低pH下由牛磷脂酰肌醇-3 ' -激酶r -亚基的SH3结构域形成的原纤维。原纤维与其他物种的分离，特别是大的聚集体，很难通过普通的生化程序实现。这里开发的方法是基于由于疾病而沉积在组织中的淀粉样原纤维可能具有高度的蛋白酶抗性这一知识。两种SH3聚集体的样品，第一种主要含有无定形聚集体，第二种主要含有淀粉样原纤维，在低pH下制备。5然后将这些样品暴露于胃蛋白酶（一种在这种条件下具有高活性的蛋白酶）中不同的时间，并通过FTIR监测消化程度（图1）。在无定形聚集体中，与β片结构（分别为1612 cm-1和1684 cm-1）相关的光谱特征迅速消失，在约1649 cm-1处留下非结构化物质的特征带。相比之下，含有淀粉样蛋白原纤维的样品，即使在3小时的孵育后，外观变化也要小得多。7有趣的是，在后一种样品中发生的变化中，我们再次看到约1684 cm-1的波段损失，而其余的聚集成分基本保持完整，仅检测到以约1618 cm-1为中心的主峰向更高波数的小位移。这些变化表明，样品中存在的无定形物质被消化后只留下抗蛋白酶原纤维。消化后在约1640-1660 cm-1处的强度增加再次归因于更多的无序物种，如肽片段。电镜观察胃蛋白酶消化过程中淀粉样蛋白原纤维的形态变化。数据显示，胃蛋白酶消化后保留了大量完整的原纤维，与原纤维在原始制剂中的整体外观相同（图2a）。原纤维内蛋白质分子的完整性通过多种方式得到证实。在6 M GndHCl中，原纤维被破坏，蛋白质被重新折叠后，记录了1D-NMR谱；光谱与天然蛋白聚集前的光谱难以区分。9对胃蛋白酶处理前后含有原纤维的样品进行SDS-PAGE分析，并随后进行超离心，结果显示，在这两种情况下，原纤维都在与SH3单体相同的位置上迁移，再次表明原纤维含有完整的全长蛋白（图2b）。胃蛋白酶-超离心后的上清液
Amyloid fibrils are highly organized proteinaceous aggregates associated with pathogenic conditions such as Alzheimer’s disease and the spongiform encephalopathies. 1 They are also being explored as novel nanostructures with a wide variety of potential applications. 2 Despite the considerable advances in the characterization of such fibrils3, 4 there are still a number of important aspects of their structure that remain to be clarified. Among these are the detailed definition of the conformational state of the protein chains within an amyloid fibril and the manner in which the constituent protofilaments are assembled. Studies to investigate such issues are hindered by the intrinsic heterogeneity of fibril samples prepared in vitro, 4 as these invariably contain soluble precursors and nonfibrillar aggregates. 5 The development of strategies to isolate fibrils is therefore of great importance. Here we report a method to achieve this objective and use it to characterize the fibrils formed at low pH by the SH3 domain of the R-subunit of bovine phosphatidylinositol-3′-kinase. Separation of fibrils from other species, particularly large aggregates, is difficult to achieve by common biochemical procedures. The method developed here was based on the knowledge that amyloid fibrils deposited in tissue as a result of disease can be highly protease resistant. 6 Samples of two types of SH3 aggregates, the first containing predominantly amorphous aggregates and the second predominantly amyloid fibrils, were prepared at low pH as described elsewhere. 5 These samples were then exposed to pepsin, a protease with high activity under such conditions, for different periods of time and the extent of digestion was monitored by FTIR (Figure 1). In amorphous aggregates spectral features associated with β-sheet structure (1612 and 1684 cm-1 respectively) disappear rapidly leaving a band at ca. 1649 cm-1 characteristic of unstructured species. Samples containing amyloid fibrils, by contrast, experience much smaller changes in appearance even after 3 h of incubation. 7 Interestingly, among the changes occurring in the latter samples again we see the loss of the band at ca. 1684 cm-1 whereas the rest of the aggregation components remain largely intact and only a small shift of the main peak, centered at ca. 1618 cm-1, to higher wavenumbers is detected. These changes suggest that the amorphous species present in the sample are digested to leave just the proteaseresistant fibrils. The increase in intensity at ca. 1640-1660 cm-1 after digestion is again attributable to a higher population of disordered species, such as peptide fragments. Morphological changes in the amyloid fibrils during pepsin digestion were analyzed by electron microscopy. The data show that abundant intact fibrils remain after pepsin digestion with the overall appearance of the fibrils in the original preparations (Figure 2a). The integrity of the protein molecules within the fibrils was confirmed in a variety of ways. 1D-NMR spectra were recorded after the fibrils had been disrupted in 6 M GndHCl and the protein refolded; the spectra were indistinguishable from those of the native protein prior to aggregation. 9 SDS-PAGE analysis of samples containing fibrils before and after treatment with pepsin and subsequent ultra-centrifugation shows in both cases a band migrating at the same position as the SH3 monomer, again indicating that the fibrils contain intact full-length protein (Figure 2b). The supernatant obtained after ultra-centrifugation of pepsin-