Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark; Sino-Danish Center (SDC), Eastern Yanqihu campus, University of Chinese Academy of Sciences, 380 Huaibeizhuang, Huairou district, Beijing.
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark; Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark.
J Mol Biol. 2022 Jan 30;434(2):167337. doi: 10.1016/j.jmb.2021.167337. Epub 2021 Nov 5.
Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.
功能性细菌淀粉样蛋白（FuBA）是无规卷曲的蛋白质（IDP），它们能快速有效地聚集，形成极其稳定的纤维。从 IDP 到淀粉样蛋白的转化是进化优化的，可能将折叠与聚集偶联。许多 FuBA 包含几个不完美的重复序列，这些序列有助于成熟 FuBA 纤维的稳定性。聚集可以被认为是蛋白质折叠过程的分子间延伸，传统上使用化学变性剂来研究该过程。在这里，我们使用变性剂来研究 CsgA 和 FapC 纤维化过程中的折叠步骤。我们根据纤维形成曲线确定的变性剂浓度依赖性（m 值）来量化纤维形成过程中不同阶段的蛋白质紧凑化（即蛋白质聚集时原本暴露的表面积的埋藏程度）。对于这两种蛋白质，尿素主要影响成核和延伸（不影响断裂），这与这些步骤涉及分子内和分子间聚集的事实一致。这两个步骤具有相似的 m 值，表明成核和延伸中的激活步骤涉及相同程度的折叠。令人惊讶的是，FapC 缺失两个或三个重复会导致纤维生长的激活步骤中的 m 值（即更高的紧凑化）更大。通过对纤维的小角 X 射线散射（SAXS）分析扩展了这一观察结果，该分析表明，重复缺失削弱了淀粉样蛋白核心，导致蛋白质中通常无结构区域的更大部分被纳入淀粉样蛋白骨架。我们得出结论，变性剂对纤维化的敏感性可以为聚集的分子机制提供有用的见解。
