Deciphering protein stability in cells.
Deciphering protein stability in cells.
复制标题
破译细胞中蛋白质的稳定性。
DOI：
10.1016/j.jmb.2013.10.004
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发表时间：
2014
影响因子：
5.6
通讯作者：
Gershenson,Anne
中科院分区：
文献类型：
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作者：
Gershenson,Anne
蛋白质已经进化到在与实验室实验中经常使用的稀释溶液完全不同的环境中折叠和起作用。细胞是拥挤的环境，含有200毫克/毫升的生物分子>，甚至细胞外环境如血浆可含有80毫克/毫升的蛋白质>。这些条件会影响蛋白质的稳定性和构象分布，促进五元结构，生物分子之间的瞬时相互作用，即粘性[2-6]。近年来，包括核磁共振[7]、拉曼显微镜[8]和荧光显微镜在内的许多方法已被用于监测细胞中的蛋白质结构、稳定性、合成和/或折叠。其中一种方法是Gruebele及其同事开发的FReI（快速松弛成像），它可以在高空间分辨率下实时测量活细胞中的蛋白质热稳定性和折叠动力学[9-11]。这是通过结合电池内测量Förster共振能量传递与小的瞬态温度跳变来实现的。这些实验的主力蛋白是酵母PGK（磷酸甘油酸激酶），这是糖酵解途径中的一种细胞质酶，其Förster共振能量转移对由供体（绿色荧光蛋白）AcGFP1 （n端）和受体（红色荧光蛋白）mCherry （c端）提供。在这一期中，Guzman等人将细胞内FReI应用于莱姆病螺旋体伯氏疏螺旋体的可变表面抗原蛋白VlsE 0。
Proteins have evolved to fold and function in environments quite different from the dilute solutions often used in laboratory experiments. Cells are crowded environments containing> 200 mg/ml of biomolecules, and even extracellular environments such as plasma can contain 80 mg/ml of protein [1]. These conditions can affect protein stability and conformational distributions and promote quinary structure, transient interactions between biomolecules, that is, stickiness [2–6].In recent years, a number of methods including NMR [7], Raman microscopy [8] and fluorescence microscopy have been adapted to monitor protein structure, stability, synthesis and/or folding in cells. One method, FReI (fast relaxation imaging) developed by Gruebele and colleagues, allows real-time measurements of protein thermal stability and folding kinetics in living cells with high spatial resolution [9–11]. This is accomplished by combining in-cell measurements of Förster resonance energy transfer with small, transient temperature jumps. The workhorse protein for these experiments has been yeast PGK (phosphoglycerate kinase), a cytoplasmic enzyme in the glycolysis pathway with the Förster resonance energy transfer pair provided by the donor, green fluorescent protein, AcGFP1 at the N-terminus and the acceptor, red fluorescent protein, mCherry at the C-terminus. In this issue, Guzman et al. apply in-cell FReI to the variable surface antigen protein VlsE from the spirochete responsible for Lyme disease Borrelia burgdorferi [12].