Arauz Edwin, Aggarwal Vasudha, Jain Ankur, Ha Taekjip, Chen Jie
Howard Hughes Medical Institute, Baltimore, Maryland 21205, United States.
Anal Chem. 2016 Apr 19;88(8):4269-76. doi: 10.1021/acs.analchem.5b04127. Epub 2016 Apr 1.
Recognition of signaling phospholipids by proteins is a critical requirement for the targeting and initiation of many signaling cascades. Most biophysical methods for measuring protein interactions with signaling phospholipids use purified proteins, which do not take into account the effect of post-translational modifications and other cellular components on these interactions. To potentially circumvent these problems, we have developed a single-molecule fluorescence approach to analyzing lipid-protein interactions in crude cell extracts. As a proof of principle for this assay, we show that a variety of lipid-binding domains (LBDs) can be recruited from cell lysates specifically onto their target phospholipids. With single-molecule analysis in real-time, our assay allows direct determination of binding kinetics for transient lipid-protein interactions and has revealed unique assembly properties and multiple binding modes of different LBDs. Whereas single-copy LBDs display transient interaction with lipid vesicles, tandem-repeat LBDs, often used as lipid biosensors, tend to form stable interactions that accumulate over time. We have extended the assay to study a cellular protein, Akt, and discovered marked differences in the lipid binding properties of the full-length protein compared to its PH domain. Importantly, we have found that phosphorylation of Akt at T308 and S473 does not affect the lipid binding behaviors of Akt, contrary to the long-standing model of Akt regulation. Overall, this work establishes the single-molecule lipid pulldown assay as a simple and highly sensitive approach to interrogating lipid-protein interactions in a setting that at least partly mimics the cellular environment.
蛋白质对信号磷脂的识别是许多信号级联反应靶向和启动的关键要求。大多数用于测量蛋白质与信号磷脂相互作用的生物物理方法使用纯化的蛋白质，而没有考虑翻译后修饰和其他细胞成分对这些相互作用的影响。为了潜在地规避这些问题，我们开发了一种单分子荧光方法来分析粗细胞提取物中的脂-蛋白相互作用。作为该检测方法的原理验证，我们表明可以从细胞裂解物中特异性地募集多种脂质结合结构域（LBD）到它们的靶磷脂上。通过实时单分子分析，我们的检测方法可以直接测定瞬时脂-蛋白相互作用的结合动力学，并揭示了不同LBD的独特组装特性和多种结合模式。单拷贝LBD与脂质囊泡显示瞬时相互作用，而经常用作脂质生物传感器的串联重复LBD倾向于形成随时间积累的稳定相互作用。我们将该检测方法扩展到研究一种细胞蛋白Akt，并发现全长蛋白与其PH结构域相比在脂质结合特性上存在显著差异。重要的是，我们发现Akt在T308和S473处的磷酸化并不影响Akt的脂质结合行为，这与长期以来的Akt调节模型相反。总体而言，这项工作确立了单分子脂质下拉检测方法是一种简单且高度灵敏的方法，可在至少部分模拟细胞环境的条件下研究脂-蛋白相互作用。
