Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect β-strand repeating units (labeled R1-R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

中文翻译：

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通过氢-氘交换和质谱法了解卷曲淀粉样蛋白聚集

Curli 生物膜内的细菌不受环境压力（例如消毒剂、抗生素）的影响，这会导致顽固性感染。了解 Curli 的主要蛋白质成分 CsgA 的聚集可能会揭示疾病相关的淀粉样蛋白生成机制。在这里，我们报告了脉冲氢-氘交换和质谱 (HDX-MS) 在研究 CsgA 聚集方面的应用，从而获得特定区域的信息。通过跟踪时间依赖性肽信号消耗，大概是不溶性原纤维形成的结果，我们获得了特定于蛋白质区域（区域特异性）的 S 形曲线。这些信号消耗曲线不仅提供了替代的聚集测量，而且还提供了对聚集中可溶性物质的洞察。HDX 数据呈现为双峰同位素分布，一个代表高度无序的物种，而另一个代表结构良好的物种。尽管这两个物种的氘吸收程度随时间保持不变，但质量较低、交换较少的物种的相对丰度以区域特定的方式增加。相同的特定于区域的聚合属性也适用于不同的聚合条件。尽管 CsgA 是一种本质上无序的蛋白质，但在原纤维中，它被认为由五个不完美的 β 链重复单元（标记为 R1-R5）组成。我们发现外部重复单元 R1 和 R5 比内部单元 R2、R3 和 R4 具有更高的聚集倾向。我们还使用 TEM 来获得结构良好的物种的补充信息。