...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy
【24h】

Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy

机译：用荧光波动光谱法测定活细胞中蛋白质杂合物的化学计量

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The brightness of fluorescence fluctuations provides information about protein interactions in the intercellular environment under equilibrium conditions. Here we demonstrate that the stoichiometry of a protein complex containing two proteins labeled with CFP and YFP can be determined by brightness analysis. The brightness profile, which characterizes the brightness as a function of the labeled protein coexpression ratio, together with brightness titra-tion experiments, provides sufficient information to quantify the composition of a protein complex under stoichiometric binding conditions. The selective and simultaneous excitation of proteins labeled with CFP and YFP by choosing different excitation wavelengths is used to identify the composition of the protein complex. Interactions between nuclear receptors and their coactivators play a crucial role in the regulation of gene expression. We choose the ligand-binding domain of retinoic X receptor and the nuclear receptor interacting domain of the steroid receptor coactivator-1 as a model for exploring the formation of a hetero-oligomer by brightness analysis directly in living cells. Our results show the formation of a heterotetramer with three nuclear receptors binding to the coactivator domain. Elimination of one of the nuclear receptor binding sites through a truncation mutant changed the interaction between both proteins significantly and led to a nuclear receptor-induced oligomerization of the truncated coactivator. Quantifying protein interactions in a cell is an important step in understanding cellular function on a molecular level. This study provides proof-of-principle experiments that illustrate the potential of brightness analysis as a powerful tool for quantifying protein interactions in living cells.
机译：荧光波动的亮度提供了有关平衡条件下细胞间环境中蛋白质相互作用的信息。在这里，我们证明了可以通过亮度分析确定包含两种被CFP和YFP标记的蛋白质的蛋白质复合物的化学计量。将亮度表征为标记的蛋白质共表达比例的函数的亮度分布图，以及亮度滴定实验，提供了足够的信息来定量化学计量结合条件下蛋白质复合物的组成。通过选择不同的激发波长来选择性和同时激发被CFP和YFP标记的蛋白质，可用于鉴定蛋白质复合物的组成。核受体及其共激活因子之间的相互作用在调节基因表达中起着至关重要的作用。我们选择视黄酸X受体的配体结合域和类固醇受体coactivator-1的核受体相互作用域作为模型，通过亮度分析直接在活细胞中探索杂合寡聚物的形成。我们的结果表明形成了具有与共激活域结合的三个核受体的异四聚体。通过截短突变体消除核受体结合位点之一显着改变了两种蛋白质之间的相互作用，并导致了核受体诱导的截短的共激活子的寡聚。定量细胞中的蛋白质相互作用是从分子水平理解细胞功能的重要步骤。这项研究提供了原理验证实验，这些实验说明了亮度分析作为量化活细胞中蛋白质相互作用的强大工具的潜力。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号