α-Synuclein plays an important role in synaptic functions by interacting with synaptic vesicle membrane, while its oligomers and fibrils are associated with several neurodegenerative diseases. The specific monomer structures that promote its membrane binding and self-association remain elusive due to its transient nature as an intrinsically disordered protein. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

α-突触核蛋白通过与突触小泡膜相互作用在突触功能中发挥重要作用，而其寡聚物和原纤维与多种神经退行性疾病有关。由于其作为本质上无序的蛋白质的瞬时性质，促进其膜结合和自缔合的特定单体结构仍然难以捉摸。在这里，我们使用批量时间分辨福斯特共振能量转移的染料间距离分布作为离散分子动力学模拟中的约束来绘制 α-突触核蛋白单体的构象空间。我们利用远紫外圆二色性和交联质谱法进一步证实了在正交实验中生成的构象系综。单分子蛋白质诱导的荧光增强测量表明，在这个构象集合中，α-突触核蛋白的一些构象出人意料地稳定，表现出比毫秒慢的构象转变。我们对构象整体的综合分析揭示了促进其在膜相互作用或低聚物和原纤维形成中的各种功能的基本结构特性和潜在构象。