Kimbrough Hannah, Jensen Jacob, Weber Caleb, Miller Tayla, Maddera Lucinda E, Babu Vignesh, Redwine William B, Halfmann Randal
Stowers Institute for Medical Research, Kansas City, MO.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA.
bioRxiv. 2025 Jan 2:2025.01.01.631016. doi: 10.1101/2025.01.01.631016.
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 DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether and thereby recruit candidate modifier proteins to mEos3-fused query proteins. This accessorization allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valency of mEosNb-fused modifiers. We show that our system recapitulates known effects of multivalency on liquid-liquid phase separation and can discriminate between nucleation mechanisms of amyloid and amyloid-like assemblies. 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。在此，我们通过创建一种针对荧光蛋白mEos3的纳米抗体（mEosNb）来扩展DAmFRET技术，从而物理连接并招募候选修饰蛋白至与mEos3融合的查询蛋白。这种辅助作用使我们能够通过调节与mEosNb融合的修饰剂的表达水平和价态，快速筛选对查询蛋白相行为的影响。我们表明，我们的系统概括了多价性对液-液相分离的已知影响，并且能够区分淀粉样蛋白和类淀粉样组装的成核机制。我们的方法为探究细胞内相变机制增添了一个新的实验维度。
