Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type I integrase and LEDGF/p75
Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type I integrase and LEDGF/p75
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DOI：
10.1096/fj.04-3373fje
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发表时间：
2005-03-01
期刊：
影响因子：
4.8
通讯作者：
Engelborghs, Y
中科院分区：
文献类型：
--
作者：
Maertens, G;Vercammen, J;Engelborghs, Y
最近，我们描述了人类免疫缺陷病毒1型(HIV-1)(1)整合酶(IN)与一种细胞蛋白--晶状体上皮源性生长因子/转录共激活因子p75(LEDGF/p75)的相互作用。我们现在用荧光相关显微镜(FCM)研究了IN和LEDGF/p75这三个独立结构域的扩散行为。我们发现，不同的增强型绿色荧光蛋白(EGFP)融合蛋白在细胞内的扩散由两个不同比例的组分来描述，并且细胞核中的平均参数与细胞质中的平均参数相当。此外，我们还证明了EGFP融合的HIV-1IN和LEDGF/p75之间的特定相互作用导致了扩散系数(D)的移动。在没有显示LEDGF/p75结合的IN缺失突变体中或在使用siRNA的LEDGF/p75击倒实验中观察到相反的变化。因此，我们证明可以使用单色流式细胞术(ScFCM)在活细胞中研究蛋白质-蛋白质的相互作用。
Recently we described the interaction of human immunodeficiency virus type 1 (HIV-1)(1) integrase (IN) with a cellular protein, lens epithelium-derived growth factor/transcription coactivator p75 (LEDGF/p75). We now present the study of the diffusion behavior of the three independent domains of IN and LEDGF/p75 using fluorescence correlation microscopy (FCM). We show that diffusion in the cell of the different enhanced green fluorescent protein (EGFP) fusion proteins is described by two components with different fractions and that the average parameters in the nucleus are comparable with those in the cytoplasm. In addition, we demonstrate that specific interaction between EGFP-fused HIV-1 IN and LEDGF/p75 results in a shift in diffusion coefficient (D). The opposite shift was observed in an IN-deletion mutant that does not exhibit LEDGF/p75 binding or in a LEDGF/p75 knock-down experiment using siRNA. We thus demonstrate that protein-protein interactions can be studied in living cells, using single-color FCM (scFCM).