Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation
Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation
复制标题
DOI：
10.1073/pnas.0812227106
复制
发表时间：
2009-05-12
影响因子：
11.1
通讯作者：
Dobson, Christopher M.
中科院分区：
文献类型：
--
作者：
Carullaa, Natalia;Zhou, Min;Dobson, Christopher M.
最近的实验证据表明，淀粉样蛋白原纤维形成过程中聚集的中间体是导致阿尔茨海默病和II型糖尿病等日益常见的疾病发展的主要毒性部分。我们在这里描述的应用程序的脉冲标记氢氘（HD）交换策略监测质谱（MS）和核磁共振光谱（NMR）表征的聚集过程中的SH3域在2个不同的条件下，这两个最终导致定义明确的淀粉样纤维。在一种条件下，中间体似乎在本质上基本上是无定形的，而在另一种条件下，原纤维物种是明显的。在有利于无定形样中间体的条件下，除了显示出高度保护的成熟原纤维之外，仅可以检测到对HD交换没有保护的物种。相比之下，在有利于原纤维样中间体的条件下，MS揭示了多个物种存在不同程度的HD交换保护，表明聚集最初通过相对无序的物种发生，随后演变为形成有序的聚集体，最终导致淀粉样蛋白原纤维。使用NMR的进一步分析提供了关于聚集期间发生的结构重组以及它们发生的时间尺度的残留物特异性信息。
Recent experimental evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addition to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further analysis using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur.