使用基因编码的红外探针追踪G蛋白结合受体激活
Shixin Ye1, Ekaterina Zaitseva, Gianluigi Caltabiano
1Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
Nature
|April 13, 2010
概括
研究人员发现了Rhodopsin激活中的早期构造变化. 这些G蛋白结合受体 (GPCR) 运动先于主要螺旋转移,为受体功能提供了洞察力.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 结构生物学 结构生物学
背景情况:
- 罗多普辛是一种G蛋白结合受体 (GPCR),调解暗光视力.
- 光诱导的视网膜异质化通过跨膜螺旋的重新排列触发了罗多素的激活.
- 这些螺旋运动的确切时间和机制仍然不完全理解.
研究的目的:
- 为了阐明罗多素激活过程中形状变化的时间序列.
- 为了研究在G蛋白结合和受体激活之前的早期分子事件.
主要方法:
- 局部导向的突变发生,将p-azido-l-phenylalanine纳入罗多普辛.
- 红外光谱学用于监测阿齐多探测器的振动信号.
- 在整个罗多素激活途径中跟踪构造变化.
主要成果:
- 在不活跃的Meta I状态中检测到探头静电环境的显著变化.
- 早期的变化表明H6旋转和H5运动在活跃的Meta II状态之前.
- H6的明显向外倾斜定义了活跃的Meta II状态,打开了细胞质表面.
结论:
- 在罗多素激活的早期发生了形状变化,先于主要的螺旋结构重组.
- 这些发现为解释GPCR晶体结构提供了一个框架.
- 这项研究揭示了其他GPCRs激活期间的构造子状态.
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G-protein Coupled Receptors
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
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Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
