应用太阳成集团tyc122ccvip的荧光相关光谱单分子分析仪研究新冠病毒入侵人体细胞的分子机制
发布时间：
2023-05-06
分类：
美国麻省大学医学院的James Munro团队应用单分子荧光共振能量转移（smFRET）和荧光相关光谱（FCS）技术揭示了新冠病毒(SARS-CoV-2)入侵呼吸道上皮细胞的分子机制。smFRET实验表明，SARS-CoV-2突刺蛋白（S蛋白）的受体结合域(RBD)存在两种构象：“up”和“down”；RBD处于up构象时，更有利于结合细胞膜表面的血管紧张素转换酶2(ACE2)受体，从而入侵细胞。通过smFRET实验，发现RBD与ACE2结合降低了前者的up构象到down构象的转换速率，从而稳定了RBD的up构象，更有利于病毒入侵。作者还对比了变异毒株D614G和原始毒株D614的smFRET实验结果，发现D614G的RBD处于up构象的占比更高，这解释了变异毒株更容易入侵人体的一个原因。此外，作者研究了多种单克隆抗体和D614、D614G病毒株S蛋白结合前后的RBD构象变化，发现与S蛋白颈部（stalk region）结合的抗体可稳定RBD的up构象，并暴露与ACE2受体的结合位点。应用太阳成集团tyc122ccvip开发的桌面式荧光相关光谱仪（CorTector SX系列），该研究团队测量了D614、D614G病毒株的S蛋白与ACE2受体结合反应的亲和力（KD值），以及多种单克隆抗体对这一亲和力的调控作用。这些FCS数据支持了RBD的up构象更有利于ACE2受体结合的假说，并对“鸡尾酒”抗体疗法提供了新思路：即应用与S蛋白颈部结合的抗体去促进RBD的up构象，从而更有利于RBD特异性抗体去阻断ACE2受体结合。
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Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging, we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and in the presence of the D614G mutation. We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails. |
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