Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.

细胞内大分子拥挤会导致蛋白质聚集增加，尤其是那些缺乏天然折叠构象的蛋白质。拥挤也可以通过在体外添加聚乙二醇（PEG）等聚合物来模拟。 α-突触核蛋白是一种本质上无序的蛋白质，在拥挤的环境中表现出聚集和淀粉样原纤维形成增加。人们提出了两个假设来解释这一观察结果。一是排除体积效应，认为拥挤环境中水分活度的降低会导致有效蛋白质浓度增加，从而促进聚集。另一种解释是，拥挤的增加促进了向非本地形式的转换，从而提高了聚集率。在这项工作中，我们将这两种假设分开，以研究哪一种假设正在发挥作用。我们表明，仅仅增加 α-突触核蛋白的浓度不足以诱导聚集和随后的纤维化。在体外，我们发现 PEG 浓度和聚集之间存在复杂的关系，其中较小的 PEG 会延迟纤维颤动；而较大的则促进原纤维成核。反过来，虽然 PEG600 没有增加聚集速率，但 PEG1000 却增加了聚集速率，而 PEG4000 和 PEG12000 则减慢了聚集速率，但导致后者的病例总体纤维负担更高。在细胞中，PEG4000 可减少 α-突触核蛋白的聚集，但其方式因细胞环境/细胞因素而异。当与 PEG8000 或 PEG12000 处于相同浓度时，类似大小的球状溶菌酶的聚集在体外不会增加。因此，天然无序的 α-突触核蛋白在特定类型的拥挤环境中经历构象转变，形成易于聚集的构象异构体。