Protein liquid–liquid phase separation can lead to disease-related amyloid fibril formation. The mechanisms of conversion of monomeric protein into condensate droplets and of the latter into fibrils remain elusive. Here, using mass photometry, we demonstrate that the Parkinson’s disease-related protein, α-synuclein, can form dynamic nanoscale clusters at physiologically relevant, sub-saturated concentrations. Nanoclusters nucleate in bulk solution and promote amyloid fibril formation of the dilute-phase monomers upon ageing. Their formation is instantaneous, even under conditions where macroscopic assemblies appear only after several days. The slow growth of the nanoclusters can be attributed to a kinetic barrier, probably due to an interfacial penalty from the charged C terminus of α-synuclein. Our findings reveal that α-synuclein phase separation occurs at much wider ranges of solution conditions than reported so far. Importantly, we establish mass photometry as a promising methodology to detect and quantify nanoscale precursors of phase separation. We also demonstrate its general applicability by probing the existence of nanoclusters of a non-amyloidogenic protein, Ddx4n1.

蛋白质液-液相分离可导致与疾病相关的淀粉样原纤维形成。单体蛋白质转化为冷凝液滴以及后者转化为原纤维的机制仍然难以捉摸。在这里，我们使用质量光度测定法证明，帕金森病相关蛋白 α-突触核蛋白可以在生理相关的亚饱和浓度下形成动态纳米级簇。纳米簇在本体溶液中成核并促进老化时稀相单体的淀粉样原纤维形成。即使在几天后才出现宏观组装的情况下，它们的形成也是瞬时的。纳米簇的缓慢生长可归因于动力学势垒，这可能是由于 α-突触核蛋白带电 C 末端的界面惩罚。我们的研究结果表明，α-突触核蛋白相分离发生的溶液条件范围比迄今为止报道的要宽得多。重要的是，我们将质量光度测定法建立为一种有前途的方法来检测和量化相分离的纳米级前体。我们还通过探测非淀粉样蛋白 Ddx4n1 纳米簇的存在来证明其普遍适用性。