Fang Xingru, Cao Xurui, Zhang Xiaotong, Li Qi, Huang Wenwen, Zhang Yu, Liu Honglin
Joint Research Center for Food Derived Functional Factors and Synthetic Biology of IHM, Anhui Provincial International Science and Technology Cooperation Base for Major Metabolic Diseases and Nutritional Interventions, China Light Industry Key Laboratory of Meat Microbial Control and Utilization, School of Food and Biological Engineering, Engineering Research Center of Bio-process, Ministry of Education, School of Chemistry and Chemical Engineering, Hefei University of Technology Hefei 230601 P. R. China
Chem Sci. 2025 Jul 29. doi: 10.1039/d5sc02717a.
Nanoscale dimerization geometries dictate transmembrane receptor phosphorylation dynamics, yet current methods fail to directly correlate spatial receptor organization with real-time phosphorylation states . Receptor tyrosine kinase (RTK) activation, essential for cellular signaling, is dynamically controlled by dimerization at the nanoscale, but existing tools cannot simultaneously resolve spatial dimer configurations and phosphorylation activity. Here, we developed a transmembrane nanogold surface energy transfer ruler (T-Nanoruler) to enable tracking of RTK activation. This system integrates extracellular DNA aptamers (ss--FAM) that program receptor dimerization spacer (0-8.6 nm) with intracellular gold nanoparticle-antibody conjugates (G@P-Met) targeting phosphorylated residues, creating a molecular ruler for nanoscale distance quantification. Using Met as a model, flow cytometry revealed distance-dependent quenching efficiencies that precisely matched phosphorylation levels validated by western blotting and scratch wound assays. Tight dimerization (0 nm spacer) maximized phosphorylation and cell migration, whereas extended configurations (8.6 nm spacer) suppressed activation. T-Nanoruler overcomes the limitations of FRET (limited detection range) and conventional methods (poor spatiotemporal resolution), establishing a versatile framework for probing spatial regulation across diverse RTK signaling systems. Our results established the first quantitative paradigm for synchronously mapping phosphorylation dynamics and dimerization geometries with nanometer precision, offering unprecedented insights into allosteric transmembrane signaling mechanisms.
纳米级二聚化几何结构决定跨膜受体磷酸化动力学，但目前的方法未能直接将空间受体组织与实时磷酸化状态相关联。受体酪氨酸激酶（RTK）激活是细胞信号传导所必需的，它在纳米尺度上通过二聚化受到动态控制，但现有工具无法同时解析空间二聚体构型和磷酸化活性。在此，我们开发了一种跨膜纳米金表面能量转移尺（T-Nanoruler）以实现对RTK激活的追踪。该系统整合了细胞外DNA适配体（ss-FAM），其对受体二聚化间隔区（0 - 8.6纳米）进行编程，以及针对磷酸化残基的细胞内金纳米颗粒 - 抗体偶联物（G@P-Met），创建了一个用于纳米级距离定量的分子尺。以Met为模型，流式细胞术揭示了与距离相关的淬灭效率，其与通过蛋白质印迹和划痕伤口试验验证的磷酸化水平精确匹配。紧密二聚化（0纳米间隔区）使磷酸化和细胞迁移最大化，而扩展构型（8.6纳米间隔区）则抑制激活。T-Nanoruler克服了荧光共振能量转移（检测范围有限）和传统方法（时空分辨率差）的局限性，建立了一个用于探测各种RTK信号系统中空间调节的通用框架。我们的结果建立了第一个以纳米精度同步绘制磷酸化动力学和二聚化几何结构的定量范式，为变构跨膜信号传导机制提供了前所未有的见解。
