Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction.
Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction.
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DOI：
10.1016/j.chembiol.2017.06.015
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发表时间：
2017-08-17
影响因子：
8.6
通讯作者：
Howarth M
中科院分区：
文献类型：
--
作者：
Jacobsen MT;Fairhead M;Fogelstrand P;Howarth M
蛋白质的化学修饰为控制和可视化生命系统提供了绝佳的机会。修饰蛋白质最常见的方法是将其丰富的胺与 N-羟基琥珀酰亚胺 (NHS) 酯反应。在这里，我们使用核心研究工具链霉亲和素-生物素探讨胺数和位置对蛋白质缀合物行为的影响。链霉亲和素的染料 NHS 修饰严重破坏了配体结合，因此需要开发一种新的链霉亲和素，在标记后保持超稳定的结合。为了探索理想的染料改性水平，我们设计了一个每个亚基含有 1-6 个胺的面板：“胺美化”。令人惊讶的是，亮度随着胺数的减少而增加，揭示了传统标记后的广泛淬灭。我们最终选择了 Flavidin（荧光团友好的链霉亲和素），它将超稳定的配体结合与缀合后增加的亮度结合在一起。黄素增强了荧光成像，使组织中的细胞标记更加灵敏和特异。黄素在分子检测中应该有广泛的应用，为如何同时优化生物分子和化学探针的行为提供一般见解。用小分子染料标记链霉亲和素会损害配体结合 K121R 突变可在染料标记后挽救配体稳定性 蛋白质胺的美化可优化亮度 荧光团友好的链霉亲和素可提高成像特异性和灵敏度 染料的化学偶联经常会损害蛋白质功能。雅各布森等人。使用广泛使用的工具链霉亲和素探索了这一挑战。通过胺景观策略，他们生产了“荧光团友好的链霉亲和素”，它将极端的配体结合稳定性与更明亮、更特异的细胞成像性能结合起来。
Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1–6 amines per subunit: “amine landscaping.” Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe. Labeling of streptavidin with small-molecule dyes impairs ligand binding K121R mutation rescues ligand stability after dye labeling Landscaping of protein amines optimizes brightness Fluorophore-friendly streptavidin improves imaging specificity and sensitivity Chemical coupling of dyes frequently damages protein function. Jacobsen et al. explored this challenge using the widespread tool streptavidin. Through an amine-landscaping strategy, they produced “Fluorophore-friendly streptavidin” which combines extreme ligand-binding stability with brighter and more specific cellular imaging performance.
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影响因子：
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作者：
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通讯作者：
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通讯作者：
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