Structure and dynamics of a partially folded protein are decoupled from its mechanism of aggregation
Structure and dynamics of a partially folded protein are decoupled from its mechanism of aggregation
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DOI：
10.1021/ja8029224
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发表时间：
2008-10-01
影响因子：
15
通讯作者：
Chiti, Fabrizio
中科院分区：
文献类型：
--
作者：
Calloni, Giulia;Lendel, Christofer;Chiti, Fabrizio
研究淀粉样蛋白形成机制的常用策略是表征前体状态的结构和动力学，在大多数情况下，前体状态是部分折叠的蛋白质。在这里，我们研究了高度动态的构象状态形成的蛋白质结构域HypF-N在低pH值下，聚集前，使用荧光，圆二色性，和NMR光谱。NMR分析使我们，特别是，以确定区域的序列，形成疏水相互作用，并采用α-螺旋二级结构的pH变性的合奏。为了理解这种残留结构在这种蛋白质的聚集中所起的作用，我们使用蛋白质工程实验探索了聚集的机制，从而确定了HypF-N序列的区域，这些区域在将这种动态状态转化为硫磺素T结合和含有β-折叠的原纤维中起关键作用。这两种互补方法的组合揭示了pH变性的HypF-N的聚集不是结构依赖性的，这意味着它不是由蛋白质的区域驱动的，这些区域在初始部分折叠状态下受到较少或较多的保护。相反，它是由离散的蛋白质区域促进的，这些蛋白质区域由于其物理化学性质而具有最高的内在聚集倾向。
A common strategy to study the mechanism of amyloid formation is the characterization of the structure and dynamics of the precursor state, which is in most cases a partially folded protein. Here we investigated the highly dynamic conformational state formed by the protein domain HypF-N at low pH, before aggregation, using fluorescence, circular dichroism, and NMR spectroscopies. The NMR analysis allowed us, in particular, to identify the regions of the sequence that form hydrophobic interactions and adopt an a-helical secondary structure in the pH-denatured ensemble. To understand the role that this residual structure plays in the aggregation of this protein, we probed the mechanism of aggregation using protein engineering experiments and thus identified the regions of the sequence of HypF-N that play a critical role in the conversion of this dynamic state into thioflavin T-binding and beta-sheet containing protofibrils. The combination of these two complementary approaches revealed that the aggregation of pH-denatured HypF-N is not structure-dependent, meaning that it is not driven by the regions of the protein that are either less or more protected in the initial partially folded state. It is, by contrast, promoted by discrete protein regions that have the highest intrinsic propensity to aggregate because of their physicochemical properties.