Ganesan Sundar, Ameer-Beg Simon M, Ng Tony T C, Vojnovic Borivoj, Wouters Fred S
Cell Biophysics Group, European Neuroscience Institute-Göttingen, Waldweg 33, 37073 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. doi: 10.1073/pnas.0509922103. Epub 2006 Mar 6.
Förster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.
Förster共振能量转移（FRET）显微镜是一种强大的技术，能够可视化信号转导中间体、蛋白质相互作用以及蛋白质的构象和生化状态。随着荧光蛋白种类的不断增加，光谱不同的荧光蛋白变体对已被用于活细胞中FRET的研究。然而，高效FRET所需的合适光谱重叠受到发射光适当分离要求的限制。目前使用的FRET对是在这些相互矛盾的光谱需求之间做出的妥协，这降低了可达到的最大FRET灵敏度。我们提出了一种以前未描述过的FRET受体，一种名为REACh（共振能量接受型彩色蛋白）的非荧光黄色荧光蛋白（YFP）突变体。REACh允许使用光物理性能更优的FRET供体EGFP，与之表现出最佳的光谱重叠，这无需窄光谱滤波，并允许在同一细胞内使用额外的荧光标记。后者使得能够针对复杂的生物学问题生成复杂的生物测定法。我们表明，这种暗受体非常适合供体荧光寿命成像显微镜（FLIM），并通过基于强度的独立供体荧光猝灭共振能量转移（FqRET）测定法证实了这些测量结果。REACh还可用于基于供体光漂白动力学的FRET研究。通过检测绿色荧光蛋白（GFP）标记的泛素化底物与REACh标记的泛素之间的FRET，我们对细胞内活跃的泛素化机制进行了成像。因此，该测定法可用于研究其功能受泛素化调节的蛋白质。
