Single molecular analysis of protein translocation dynamics on the endoplasmic reticulum membrane

内质网膜上蛋白质易位动力学的单分子分析

• 批准号: 22770153
• 负责人: YAMAMOTO Hitoshi
• 金额: $ 2.75万
• 依托单位: University of Hyogo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2010
• 资助国家: 日本
• 起止时间: 2010 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22770153/
• 关键词: 1分子計測; 操作; 膜タンパク質; 膜透過; 一分子計測; 蛋白質; 生体膜; 膜透過ダイナミクス; 生物物理

Protein translocation and integration are essential processes by which proteins are transported to their proper destinations. These processes proceed on the endoplasmic reticulum membrane in eukaryotic cells. In this study, we aimed at single molecule analysis of protein translocation dynamics. Protein translocation intermediate were synthesized with using cell free protein synthesis system and microsome, and labeled with fluorescent dye by suppressor-tRNA methods. The fluorescence and quenching were successfully observed by using total internal reflection fluorescence microscopy. However, due to the low S/N ratio caused by high background light, low fluorescence intensity and short life time, it was found that effective fluorescence intensity change was hardly observed. Thus, improving the S/N ratio remained to be as future work.

蛋白质的转运和整合是蛋白质被运送到合适的目的地的基本过程。这些过程在真核细胞的内质网膜上进行。在这项研究中，我们旨在对蛋白质易位动力学进行单分子分析。利用无细胞蛋白质合成系统和微粒子合成蛋白质转位中间体，并用抑制子-tRNA方法标记荧光染料。用全内反射荧光显微镜成功地观察到了荧光和猝灭现象。然而，由于高背景光、低荧光强度和短寿命导致的低S/N比，几乎没有观察到有效的荧光强度变化。因此，提高S/N的比例仍然是未来的工作。

项目成果

Polypeptide movement in the protein conducting channel is modulated by cholesterol of the endoplasmic reticulum membrane

蛋白质传导通道中的多肽运动受到内质网膜胆固醇的调节

• 发表时间: 2011
• 作者: Sekimoto J;Kabayama K;Gohara K;Inokuchi JI;Yamamoto Hitoshi
• 通讯作者: Yamamoto Hitoshi