Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation
Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation
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DOI：
10.1002/cbic.202100188
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发表时间：
2021-06-09
期刊：
影响因子：
3.2
通讯作者：
Nelson, Christian D. S.
中科院分区：
文献类型：
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作者：
Baccile, Joshua A.;Voorhees, Peter J.;Nelson, Christian D. S.
JC多瘤病毒的主要衣壳蛋白VP1组装成五聚体，作为研究病毒进入这种潜在严重人类病原体的模型。以前，用荧光染料标记病毒蛋白利用大融合蛋白或非特异性胺或半胱氨酸功能化。这些立体阻碍融合蛋白或异质标记病毒粒子的成像限制了再现性，并可能阻止检测细微的运输现象。在这里，我们提出了pi夹介导的半胱氨酸偶联用于vp1五聚体的位点选择性荧光标记。我们演示了一步合成探针，该探针由生物正交点击化学手柄通过聚乙二醇连接剂桥接到全氟联苯pi钳反应性亲电试剂组成。我们通过展示VP1内部表面暴露环路的选择性标记，扩大了pi钳共轭的范围。因此，pi-clamp偶联提供了一种通用的方法，可以选择性地将感兴趣的标签与病毒蛋白生物偶联，而不会阻碍它们结合和进入细胞的能力。
The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the pi-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl pi-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the pi-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the pi-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.