The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP–substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

中文翻译：

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自标记蛋白质标签 HaloTag7、SNAP-tag 和 CLIP-tag 的动力学和结构表征

自标记蛋白质标签 (SLP) HaloTag7、SNAP-tag 和 CLIP-tag 允许用合成分子共价标记融合蛋白，用于生物成像和生物技术。为了指导 SLP-底物对的选择并为底物设计提供指导，我们报告了对 HaloTag7、SNAP-tag 和 CLIP-tag 的标记动力学和底物特异性的系统和比较研究。HaloTag7 对某些罗丹明底物几乎达到了扩散受限的标记速率常数，比对应底物的 SNAP-tag 高 2 个数量级以上。然而，与 HaloTag7 相比，SNAP-tag 标记速率常数受标签结构的影响较小，对于常用底物而言，其变化超过 6 个数量级。确定用荧光底物标记的 HaloTag7 和 SNAP-tag 的晶体结构使我们能够合理化它们的底物偏好。我们还展示了如何利用这些见解来设计具有改进标记动力学的底物。