Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies permitting us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

通过蛋白质和核酸的相分离形成的生物分子缩合物被认为与多种细胞功能和功能障碍有关。通过进行单分子研究，我们剖析了与肉瘤中本质无序的类朊病毒低复杂性融合结构域的相分离相关的关键分子事件，使我们能够获得传统整体实验中存在的大量分子信息。我们的单分子 FRET 实验揭示了两个构象不同的单体亚群的共存。单液滴单分子 FRET 研究与荧光相关光谱、皮秒时间分辨荧光各向异性和振动拉曼光谱相结合表明，结构解旋将分子内相互作用转变为分子间接触，从而在凝聚物内形成动态网络。与疾病相关的突变引入了增强的结构可塑性，从而产生更大的链间相互作用，从而加速病理聚集。我们的研究结果为序列编码的动态控制结构解压缩导致生物相分离提供了关键的机制基础。