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【Nature Methods编译】细胞环境中检测蛋白质的相互
Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells
来源
Nature Methods 2, 261 - 268 (2005)
Published online: 23 March 2005; | doi:10.1038/nmeth752
作者
Monika Suchanek, Anna Radzikowska & Christoph Thiele
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, D-01307 Dresden, Germany.
Correspondence should be addressed to Christoph Thiele thiele@mpi-cbg.de
摘要原文
Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.
原文链接
http://www.nature.com/cgi-taf/DynaPage.taf?file=/nmeth/journal/v2/n4/abs/nmeth752.html
编译
细胞环境中检测蛋白质的相互作用的唯一直接方法是光交联反应。这里，我们建立了一个活细胞中蛋白质光交联反应的新方法。研究者设计了两个新的光敏化氨基酸，命名为光化蛋氨酸和光化亮氨酸，因为它们的结构和性质都分别与自然蛋氨酸和亮氨酸极为相似。这种相似使他们逃过了氨基酸合成过程中严格的识别控制机制，并被未改变的哺乳动物翻译组件合并到蛋白质中。紫外线的活化作用诱导了蛋白质的共价交联，这可以通过简单的western blotting高度特异地检测出来。这一技术运用到膜蛋白复合物，科学家发现了一个先前未知的孕酮结合膜蛋白PGRMC1与 Insig-1（胆固醇体内平衡的主要调节器）直接相互作用。 [标签:content1][标签:content2]
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作者:admin@医学,生命科学 2011-05-06 05:14
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