Kimbrough Hannah, Jensen Jacob, Weber Caleb, Miller Tayla, Maddera Lucinda E, Blanck Jillian F, Babu Vignesh M P, Redwine William B, Halfmann Randal
Stowers Institute for Medical Research, Kansas City, Missouri, USA.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, USA.
Protein Sci. 2025 Jul;34(7):e70194. doi: 10.1002/pro.70194.
Proteins commonly self-assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each protein's sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self-assembly in cells. Here, we extend the utility of DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether other proteins to DAmFRET-enabled query proteins. This tool allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valence of mEosNb-fused modifier proteins. We use our system to identify thresholds of valence for liquid-liquid phase separation and to discriminate nucleation mechanisms of amyloid and other paracrystalline assemblies in cells. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions.
蛋白质通常会自我组装，形成具有多种生物活性的液体或固体凝聚物。组装机制由每种蛋白质的序列和细胞环境决定。我们之前开发了分布式两性荧光共振能量转移（DAmFRET）技术，用于分析细胞中自我组装的序列决定因素。在这里，我们通过创建一种针对荧光蛋白mEos3的纳米抗体（mEosNb），将其他蛋白质物理连接到具有DAmFRET功能的查询蛋白上，从而扩展了DAmFRET的应用。该工具使我们能够通过调节与mEosNb融合的修饰蛋白的表达水平和价态，快速筛选对查询蛋白相行为的影响。我们利用该系统确定液-液相分离的价态阈值，并区分细胞中淀粉样蛋白和其他准晶体组装的成核机制。我们的方法为研究细胞内相变机制增加了一个新的实验维度。
