Krause Christopher D, Mei Erwen, Xie Junxia, Jia Yiwei, Bopp Martin A, Hochstrasser Robin M, Pestka Sidney
Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635, USA.
Mol Cell Proteomics. 2002 Oct;1(10):805-15. doi: 10.1074/mcp.m200065-mcp200.
Our experiments were designed to test the hypothesis that the cell surface interferon gamma receptor chains are preassembled rather than associated by ligand and to assess the molecular changes on ligand binding. To accomplish this, we used fluorescence resonance energy transfer, a powerful spectroscopic technique that has been used to determine molecular interactions and distances between the donor and acceptor. However, current commercial instruments do not provide sufficient sensitivity or the full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins in living cells. In our experiments, we used the blue fluorescent protein and green fluorescent protein pair, attached a monochrometer and charge-coupled device camera to a modified confocal microscope, reduced background fluorescence with the use of two-photon excitation, and focused on regions of single cells to provide clear spectra of fluorescence resonance energy transfer. In contrast to the prevailing view, the results demonstrate that the receptor chains are preassociated and that the intracellular domains move apart on binding the ligand interferon gamma. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.
我们设计了实验来检验细胞表面干扰素γ受体链是预先组装而非由配体结合在一起的这一假说，并评估配体结合时的分子变化。为实现这一点，我们使用了荧光共振能量转移，这是一种强大的光谱技术，已被用于确定供体和受体之间的分子相互作用及距离。然而，目前的商业仪器灵敏度不足，也无法提供完整光谱以得出活细胞中用蓝色和绿色荧光蛋白标记的蛋白质之间相互作用的决定性结果。在我们的实验中，我们使用了蓝色荧光蛋白和绿色荧光蛋白对，将单色仪和电荷耦合器件相机连接到一台经过改装的共聚焦显微镜上，利用双光子激发降低背景荧光，并聚焦于单细胞区域以提供清晰的荧光共振能量转移光谱。与普遍观点相反，结果表明受体链是预先结合的，并且在结合配体干扰素γ时细胞内结构域会分开。这项技术的应用应该会带来用于高通量筛选和描绘细胞相互作用组的新的快速方法。
