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APPLYING THE FLUORESCENCE CORRELATION SPECTROSCOPY SINGLE MOLECULE ANALYZER OF GUANGDONG ZHONGKE AOHUI TECHNOLOGY CO., LTD. TO STUDY THE MOLECULAR MECHANISM OF THE NEW CORONAVIRUS INVADING HUMAN CELLS

APPLYING THE FLUORESCENCE CORRELATION SPECTROSCOPY SINGLE MOLECULE ANALYZER OF GUANGDONG ZHONGKE AOHUI TECHNOLOGY CO., LTD. TO STUDY THE MOLECULAR MECHANISM OF THE NEW CORONAVIRUS INVADING HUMAN CELLS

(Summary description)James Munro 's team from the University of Massachusetts Medical School applied single-molecule fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) techniques to reveal the molecular mechanism of the new coronavirus (SARS-CoV-2) invading respiratory epithelial cells . The smFRET experiment shows that the receptor binding domain (RBD) of SARS-CoV-2 spike protein (S protein) has two conformations: "up" and "down". When the RBD is in the up conformation, it is more conducive to binding blood vessels on the surface of the cell membrane Tensin-converting enzyme 2 (ACE2) receptors, thereby invading cells. Through smFRET experiments, it was found that the combination of RBD and ACE2 reduced the conversion rate of the former up conformation to down conformation, thereby stabilizing the up conformation of RBD, which is more conducive to virus invasion. The author also compared the smFRET experiment results of the mutant strain D614G and the original strain D614, and found that the RBD of D614G has a higher proportion in the up conformation, which explains one reason why the mutant strain is more likely to invade the human body. In addition, the authors studied the conformational changes of RBD before and after the binding of a variety of monoclonal antibodies to the S protein of D614 and D614G virus strains, and found that antibodies bound to the neck of the S protein (stalk region) can stabilize the up conformation of RBD and expose the receptors associated with ACE2. body binding sites. Using the desktop fluorescence correlation spectrometer (CorTector SX series) developed by Guangdong Zhongke Aohui Technology Co., Ltd., the research team measured the affinity (K D value) of the binding reaction between the S protein of the D614 and D614G virus strains and the ACE2 receptor, and Modulation of this affinity by various monoclonal antibodies. These FCS data support the hypothesis that the up conformation of RBD is more conducive to ACE2 receptor binding, and provide a new idea for "cocktail" antibody therapy: that is, the application of antibodies that bind to the neck of the S protein to promote the up conformation of RBD, thereby more It is beneficial for RBD-specific antibodies to block ACE2 receptor binding.

APPLYING THE FLUORESCENCE CORRELATION SPECTROSCOPY SINGLE MOLECULE ANALYZER OF GUANGDONG ZHONGKE AOHUI TECHNOLOGY CO., LTD. TO STUDY THE MOLECULAR MECHANISM OF THE NEW CORONAVIRUS INVADING HUMAN CELLS

(Summary description)James Munro 's team from the University of Massachusetts Medical School applied single-molecule fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) techniques to reveal the molecular mechanism of the new coronavirus (SARS-CoV-2) invading respiratory epithelial cells . The smFRET experiment shows that the receptor binding domain (RBD) of SARS-CoV-2 spike protein (S protein) has two conformations: "up" and "down". When the RBD is in the up conformation, it is more conducive to binding blood vessels on the surface of the cell membrane Tensin-converting enzyme 2 (ACE2) receptors, thereby invading cells. Through smFRET experiments, it was found that the combination of RBD and ACE2 reduced the conversion rate of the former up conformation to down conformation, thereby stabilizing the up conformation of RBD, which is more conducive to virus invasion. The author also compared the smFRET experiment results of the mutant strain D614G and the original strain D614, and found that the RBD of D614G has a higher proportion in the up conformation, which explains one reason why the mutant strain is more likely to invade the human body. In addition, the authors studied the conformational changes of RBD before and after the binding of a variety of monoclonal antibodies to the S protein of D614 and D614G virus strains, and found that antibodies bound to the neck of the S protein (stalk region) can stabilize the up conformation of RBD and expose the receptors associated with ACE2. body binding sites. Using the desktop fluorescence correlation spectrometer (CorTector SX series) developed by Guangdong Zhongke Aohui Technology Co., Ltd., the research team measured the affinity (K D value) of the binding reaction between the S protein of the D614 and D614G virus strains and the ACE2 receptor, and Modulation of this affinity by various monoclonal antibodies. These FCS data support the hypothesis that the up conformation of RBD is more conducive to ACE2 receptor binding, and provide a new idea for "cocktail" antibody therapy: that is, the application of antibodies that bind to the neck of the S protein to promote the up conformation of RBD, thereby more It is beneficial for RBD-specific antibodies to block ACE2 receptor binding.

Information

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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.

For more details, please pay attention to the single-molecule fluorescence official account or click the link below.

https://mp.weixin.qq.com/s?__biz=MzkzNzI0NTc5Mg==&mid=2247485565&idx=1&sn=7f646fdbaec97b6ab26e396744930483&chksm=c2932572f5e4ac64b02f22a41cbad20cd 52eec287fafa8e14f62e2b8b55c839a46a9e426ea3b&token=928242522&lang=zh_CN#rd

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