监测间充质上皮转化因子（Met）的二聚化状态对于深入了解肿瘤信号传导网络至关重要。目前，Met蛋白的二聚化激活途径主要在宏观水平上研究，而单个分子水平的研究远远不够全面。本文采用单分子力学光谱技术动态研究了Met蛋白的细胞外结构域受配体肝细胞生长因子（HGF）诱导的二聚化激活。将Met蛋白固定在类生物膜上以确保其生理环境，然后通过两个Met结合寡核苷酸形成的双价探针识别Met二聚体。通过一些参数（如单峰比率、双峰比率和分离功）可以将Met蛋白的二聚态与单聚态区分开来。在HGF处理之前，大多数Met蛋白仍处于单体形式，因此力曲线中的单峰比率更高（78.8±5.2%），双峰比率更低（17.0±4.1%）。经过HGF处理，单峰比率降低至54.0±7.4%，双峰比率增加至43.2±7.3%。这是由于在Met蛋白和HGF结合后形成了二聚体。此外，HGF处理后的平均分离功增加了约两倍。考虑到Met蛋白二聚化抑制剂的研究有助于开发更强效、更安全的抑制剂以显著抑制肿瘤转移，因此利用上述平台探索了不同药物（包括抗凝药、不同抗生素和抗癌药物）对Met蛋白二聚化激活的影响。结果表明，抗凝药肝素及其类似物可以明显抑制HGF介导的Met蛋白激活，而不同的抗生素和抗癌药物对Met蛋白二聚化没有显著影响。这项工作为研究蛋白质二聚化和筛选Met蛋白二聚化抑制剂提供了平台。版权所有 © 2023 Elsevier B.V.

Monitoring the dimerization state of the mesenchymal-epithelial transition factor (Met) was essential for in-depth understanding of the tumor signal transduction network. At present, the dimerization activation pathway of Met protein was mainly studied at the macro level, while the research at the single molecule level was far from comprehensive. Herein, the dimerization activation of Met protein's extracellular domain induced by ligand hepatocyte growth factor (HGF) was dynamically studied by single-molecule force spectroscopy. Met protein was immobilized on a biomimetic lipid membrane for ensuring its physiological environment, and then the Met dimers were recognized by bivalent probe which was formed by two Met-binding aptamers. Then the dimeric state of Met protein could be distinguished from monomeric state of Met protein through some parameters, (such as unimodal ratio, bimodal ratio and separation work). The unimodal indicates the occurrence of single molecule binding event, and the bimodal represents the occurrence of double binding event (also represents the presence of Met dimer). Before HGF treatment, most of the Met protein on the lipid membrane was still in the form of monomer, so the unimodal ratio in the force curve was larger (78.8 ± 5.2%), and the bimodal ratio was smaller (17.0 ± 4.1%). After HGF treatment, the unimodal ratio decreased to 54.0 ± 7.4%, and the bimodal ratio increased to 43.2 ± 7.3%. It was due to the formation of dimers after the binding of Met protein on the fluidity lipid membrane with HGF. In addition, the average separation work increased to about 2 times after HGF treatment. Given that studies of Met protein dimerization inhibitors have contributed to the development of more potent and safe inhibitors to significantly inhibit tumor metastasis, the effects of different medicines (including anticoagulant medicines, different antibiotics and anti-cancer medicines) on the dimerization activation of Met protein were then explored by the platform described above. The results showed that anticoagulant medicines heparin and its analogs can significantly inhibit HGF-mediated Met protein activation, while different antibiotics and anticancer medicines had no significant effect on the dimerization of Met protein. This work provided a platform for studying protein dimerization as well as for screening Met protein dimerization inhibitors at the single-molecule level.Copyright © 2023 Elsevier B.V. All rights reserved.