Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction,Cell Chemical Biology

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Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction
Cell Chemical Biology ( IF 6.6 ) Pub Date : 2017-07-27 , DOI: 10.1016/j.chembiol.2017.06.015
Michael T Jacobsen ₁ , Michael Fairhead ₁ , Per Fogelstrand ₂ , Mark Howarth ₁

Affiliation

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.

中文翻译：

胺景观最大化蛋白质染料荧光和超稳定蛋白质配体相互作用

蛋白质的化学修饰为控制和可视化生命系统提供了很好的机会。修饰蛋白质的最常见方法是其丰富的胺与 N-羟基琥珀酰亚胺 (NHS) 酯的反应。在这里，我们使用核心研究工具链霉亲和素-生物素探索胺数和定位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，因此需要在标记后开发一种新的保留链霉亲和素的超稳定结合。为了探索染料改性的理想水平，我们设计了一个每个亚基含有 1-6 个胺的面板：“胺景观”。令人惊讶的是，亮度随着胺数的减少而增加，显示出常规标记后的广泛猝灭。我们最终选择了 Flavidin（对荧光团友好的链霉亲和素），结合超稳定的配体结合和结合后增加的亮度。黄素增强荧光成像，允许在组织中进行更灵敏和特异性的细胞标记。黄素应该在分子检测中具有广泛的应用，为如何同时优化生物分子和化学探针的行为提供一般性的见解。

更新日期：2017-07-28

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