Department of Chemistry, Graduate School of Science, Kobe University, Kobe 657-8501, Japan.
Department of Applied Chemistry, National Yang Ming Chiao Tung University, Hsinchu 300093, Taiwan.
Proc Natl Acad Sci U S A. 2024 Sep 24;121(39):e2402162121. doi: 10.1073/pnas.2402162121. Epub 2024 Sep 18.
Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to β-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.
液-液相分离蛋白凝聚物因其在生物现象中的关键作用而受到广泛关注。它们通常表现出高流动性和可逆性，以发挥生物功能，但偶尔也作为淀粉样纤维形成的场所。为了理解蛋白凝聚物在生物功能和发病机制中的特性，在单细胞凝聚物水平上研究它们至关重要；然而，由于缺乏适用的方法，目前这一目标难以实现。在这里，我们证明光阱能够以时空可控的方式诱导α-突触核蛋白的单一液-相凝聚物形成。在与聚乙二醇共存的条件下，在空气/溶液界面处用强聚焦近红外激光照射会形成凝聚物。荧光染料标记成像表明，光诱导的凝聚物从中心到边缘具有蛋白质浓度的梯度，这表明它是通过α-突触核蛋白聚集体的光泵浦和沿着界面的扩展来制备的。此外，拉曼光谱和硫黄素 T 荧光分析表明，连续激光照射会诱导凝聚物内部蛋白质分子的结构向富含β-折叠的结构转变，最终导致凝聚物变形，进一步形成淀粉样纤维。这些观察结果表明，光阱是研究凝聚物出现和生长的微观机制以及随后导致淀粉样纤维形成的老化过程的强大技术。
