Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of mFA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and mFA+pH. We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2–3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid.

中文翻译：

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定量甲酸变性：不完美的重复对于功能性淀粉样蛋白 FapC 的稳定性至关重要。


细菌功能性淀粉样蛋白在进化上经过优化以聚集，以至于功能性淀粉样蛋白的极端鲁棒性使得详细检查其结构-功能关系变得非常困难。先前的工作表明，功能性淀粉样蛋白对传统的化学变性剂具有抵抗力，但它们在高浓度的甲酸（FA）中溶解。然而，系统研究需要对 FA 使蛋白质变性的能力进行定量分析。由假单胞菌形成的淀粉样蛋白。蛋白质 FapC 为研究 FA 变性提供了一个极好的模型。它包含三个不完美的重复序列，逐步去除这些重复序列会减慢纤维颤动并增加聚集过程中的碎片。然而，与稳定性的联系尚不清楚。我们首先使用三种小型、球状且耐酸的蛋白质校准 FA 变性。这揭示了 FA 浓度和解折叠自由能之间的线性关系，斜率为 mFA+pH（FA 和 FA 诱导的 pH 值降低的综合贡献），以及蛋白质大小和 mFA+ 之间的稳健相关性酸碱度。然后，我们测量了由具有不同重复次数的不同 FapC 变体形成的原纤维的溶解情况，作为 FA 浓度的函数。这揭示了删除至少两个重复序列后驱动淀粉样蛋白形成的残基数量下降。随着重复序列的去除，变性的中点下降。完全去除所有重复序列会导致原纤维溶解，其 FA 浓度比含有重复序列的变体低 2-3 个数量级，这表明功能性淀粉样蛋白的稳定性需要至少一个重复序列。