Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH
Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH
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DOI：
10.1016/j.bbapap.2009.10.005
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发表时间：
2010-01-01
影响因子：
3.2
通讯作者：
Chiti, Fabrizio
中科院分区：
文献类型：
--
作者：
Ahmad, Basir;Winkelmann, Julia;Chiti, Fabrizio
多肽和蛋白质从其天然状态转化为淀粉样蛋白纤维聚集体是许多病理状况的标志，包括阿尔茨海默病和淀粉样变性。越来越多的证据表明，与成熟原纤维相反，可溶性低聚物介导细胞功能障碍，最终导致疾病发作。在这项研究中，我们探索了碱性pH溶液的能力，迄今为止，碱性pH溶液仍然相对未被探索，形成大肠杆菌蛋白HypF （HypF- n）的n端结构域的部分折叠状态，随后组装形成稳定的可溶性低聚物。结果表明，HypF-N在高pH下通过双态过程展开。通过近紫外和远紫外圆二色性、本征荧光和ans衍生荧光以及DLS表征得到的碱性未折叠态表明了单体、预熔球态的特征。有趣的是，在低浓度的TFE存在下，碱性折叠的HypF-N在高浓度下聚集成稳定的低聚物。它们能够结合淀粉样蛋白特异性染料，如刚果红、ThT和ANS，含有广泛的β -片结构，用远紫外圆二色性检测到，使用原子力显微镜分析时高度为2.0-3.9 nm。这项研究补充了我们之前的一项研究，即在低和接近中性的pH值下由相同的蛋白质在形态、结构和颜色上相似的低聚物形成，为探索这些物种在不同条件下形成机制、精确分子结构和引起细胞功能障碍的能力方面存在的细微差异提供了机会。(C) 2009 Elsevier B.V.版权所有
Conversion of peptides and proteins from their native states into amyloid fibrillar aggregates is the hallmark of a number of pathological conditions, including Alzheimer's disease and amyloidosis. Evidence is accumulating that soluble oligomers, as opposed to mature fibrils, mediate cellular dysfunction, ultimately leading to disease onset. In this study, we have explored the ability of alkaline pH solutions, which have remained relatively unexplored so far, to form a partially folded state of the N-terminal domain of the Escherichia coli protein HypF (HypF-N), which subsequently assembles to form stable soluble oligomers. Results showed that HypF-N unfolds at high pH via a two-state process. Characterization of the resulting alkaline-unfolded state by near- and far-UV circular dichroism, intrinsic and ANS-derived fluorescence and DLS indicated characteristics of a monomeric, premolten globule state. interestingly, alkaline-unfolded HypF-N aggregates, at high concentration in the presence of low concentrations of TFE, into stable oligomers. These are able to bind amyloid-specific dyes, such as Congo red, ThT, and ANS, contain extensive beta-sheet structure, as detected with far-UV circular dichroism, and have a height of 2.0-3.9 nm when analysed using atomic force microscopy. This study, which complements our previous one in which morphologically, structurally, and tinctorially similar oligomers were formed at low and nearly neutral pH values by the same protein, offers opportunities to explore the fine differences existing in the mechanism of formation of these species under different conditions, in their precise molecular structure and in their ability to cause cellular dysfunction. (C) 2009 Elsevier B.V. All rights reserved.