Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy
Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy
复制标题
DOI：
10.1042/bj20030358
复制
发表时间：
2003-05-15
影响因子：
4.1
通讯作者：
Larijani, B
中科院分区：
文献类型：
--
作者：
Calleja, V;Ameer-Beg, SM;Larijani, B
能够在不干扰生理环境的情况下检测蛋白质构象的原位变化将是理解蛋白质相互作用中发生的精确机制的重要一步。我们已经开发了一种新的方法来监测蛋白质的构象变化在完整的细胞。构建了双标记的荧光绿色荧光蛋白-黄色荧光蛋白（GFP-YFP）融合蛋白。允许利用增强受体荧光（EAF）诱导的荧光共振能量转移（FRET）。此外，一种新的融合伴侣，YFPdark，已被设计为EAF-FRET的空间位阻控制。任何构象变化都会引起FRET的变化，反过来，通过荧光寿命成像显微镜（“FLIM”）检测。蛋白激酶B（PKB）/Akt，磷酸肌醇3-激酶介导的信号传导的关键组成部分，被选为这一目的。虽然在模型中已经提出了脂质结合和磷酸化引起的PKB/Akt构象变化，但其在完整细胞中的行为并不容易处理。我们在这里报告，血小板衍生生长因子（'PDGF'）刺激NIH 3 T3细胞表达的GFP-Akt-YFP结构导致FRET在质膜的损失，因此在PKB/Akt构象的变化。我们还表明，GFP-Akt-YFP构建体完全保留了其功能完整性。这种监测原位构象变化的新方法对AGC激酶超家族的其他成员和其他蛋白质具有广泛的应用。
To be able to detect ill situ changes in protein conformation without perturbing the physiological environment would be a major step forward in understanding the precise mechanism occurring in protein interaction. We have developed a novel approach to monitoring conformational changes of proteins in intact cells. A double-labelled fluorescent green fluorescent protein-yellow fluorescent protein (GFP-YFP) fusion protein has been constructed. allowing the exploitation of enhanced-acceptor-fluorescence (EAF)-induced fluorescence resonance energy transfer (FRET). Additionally, a novel fusion partner, YFPdark, has been designed to act as a sterically hindered control for EAF-FRET. Any conformational changes will cause a variation in FRET.,which, in turn. is detected by fluorescence lifetime imaging microscopy ('FLIM'). Protein kinase B (PKB)/Akt, a key component of phosphomositide 3-kinase-mediated signalling, was selected for this purpose. Although conformational changes in PKB/Akt consequent to lipid binding and phosphorylation have been proposed in models, its behaviour in intact cells has not been tractable. We report here that platelet-derived-growth factor ('PDGF') stimulation of NIH3T3 cells expressing the GFP-Akt-YFP construct resulted in a loss of FRET at the plasma membrane and hence a change in PKB/Akt conformation. We also show that the GFP-Akt-YFP construct conserves fully its functional integrity. This novel approach of monitoring the ill situ conformational changes has broad application for other members of the AGC kinase superfamily and other proteins.