A Method to Generate and Analyze Modified Myristoylated Proteins.
A Method to Generate and Analyze Modified Myristoylated Proteins.
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一种生成和分析修饰的肉豆蔻酰化蛋白质的方法。
DOI：
10.1002/cbic.201600608
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Zhang,Qisheng
中科院分区：
文献类型：
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作者：
Gao,Huanyao;Sun,Wei;Song,Zhiquan;Yu,Yanbao;Wang,Li;Chen,Xian;Zhang,Qisheng
蛋白质的共价脂质修饰对其细胞定位和功能至关重要。工程脂质基序与生物正交化学相结合，已被用来识别细胞中的肉豆蔻酰化或棕榈酰化蛋白质。然而，修饰蛋白是否具有与内源蛋白相似的特性尚未得到很好的研究，主要是由于缺乏生成和分析纯化蛋白的方法。我们开发了一种利用代谢干扰和质谱法来生产和分析修饰的肉豆蔻酰化小 GTP 酶 ADP 核糖基化因子 1 (Arf1) 的方法。测量了这些重组蛋白结合脂质体、负载和水解 GTP 的能力，并与未修饰的肉豆蔻酰化 Arf1 进行比较。酮修饰的肉豆蔻酰化 Arf1 可以进一步用荧光团偶联肼标记，随后通过荧光成像进行可视化。该方法提供了一个有效的模型系统，可以在将脂质修饰蛋白应用于细胞系统之前表征其附加功能。
Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio‐orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP‐ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone‐modified myristoylated Arf1 could be further labeled by fluorophore‐coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid‐modified proteins with additional functions before applying them to cellular systems.