Direct observation of multiple misfolding pathways in a single prion protein molecule
Direct observation of multiple misfolding pathways in a single prion protein molecule
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DOI：
10.1073/pnas.1107736109
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发表时间：
2012-04-03
影响因子：
11.1
通讯作者：
Woodside, Michael T.
中科院分区：
文献类型：
--
作者：
Yu, Hao;Liu, Xia;Woodside, Michael T.
蛋白质错误折叠是一种普遍存在的现象，与多种疾病相关。单分子方法通过高灵敏度测量蛋白质的构象波动，为破译错误折叠机制提供了强大的工具。我们应用单分子力谱直接观察朊病毒蛋白 PrP 的错误折叠，这种蛋白质以具有感染性错误折叠状态而闻名，能够通过招募天然折叠的 PrP 来传播。通过测量高分辨率光阱中处于张力下的单个 PrP 分子的折叠轨迹，我们发现天然折叠途径仅涉及两种状态，没有证据表明部分折叠的中间体可以介导错误折叠。相反，观察到频繁但短暂的转变为非途径中间体。检测到三种不同的错误折叠途径，全部从展开状态开始。值得注意的是，错误折叠率甚至高于自然折叠率。具有较高聚集倾向的突变体 PrP 显示出一些错误折叠状态的占据增加，表明这些状态可能在聚集过程中充当中间体。这些对单个错误折叠轨迹的测量证明了单分子方法通过绘制非天然折叠途径直接表征错误折叠的能力。
Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.