Protein mobility in the cytoplasm of Escherichia coli
Protein mobility in the cytoplasm of Escherichia coli
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DOI：
10.1128/jb.181.1.197-203.1999
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发表时间：
1999-01-01
影响因子：
3.2
通讯作者：
Leibler, S
中科院分区：
文献类型：
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作者：
Elowitz, MB;Surette, MG;Leibler, S
蛋白质在细菌细胞质中的扩散速率可能限制多种细胞功能，并限制体内许多生化反应的速率。在本文中，我们报告的绿色荧光蛋白（GFP）在大肠杆菌细胞质中的表观扩散系数的非侵入性测量。这些测量以两种方式进行：通过GFP荧光的光漂白和通过GFP的红色发射荧光状态的光活化（M. B。Elowitz，M. G. Surette，P. E. Wolf，J. Stock，and S.莱布勒，Curr。7：809 - 812，1997）。GFP在E.大肠杆菌DH5 α的平均速度为7.7 ± 2.5 μ m（2）/s。由GFP和位于细胞质中的麦芽糖结合蛋白结构域组成的72 kDa融合蛋白移动较慢，D-a为2.5 +/-0.6 μ m（2）/s。此外，GFP的迁移率强烈依赖于至少两个因素：第一，在高水平的GFP表达下，D-a降低到3.6 +/-0.7 μ m（2）/s;第二，向GFP中加入由六个组氨酸残基组成的小标签，将D-a降低到4.0 +/-2.0 μ m（2）/s。因此，一个单一的有效的细胞质粘度不能解释所有的值D-a在这里报道。这些测量对理解细胞内生化网络具有意义。
The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809-812, 1997). The apparent diffusion coefficient, D-a, of GFP in E. coli DH5 alpha was found to be 7.7 +/- 2.5 mu m(2)/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with D-a of 2.5 +/- 0.6 mu m(2)/s. In addition, GFP mobility can depend strongly on at least two factors: first, D-a is reduced to 3.6 +/- 0.7 mu m(2)/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces D-a to 4.0 +/- 2.0 mu m(2)/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of D-a reported here. These measurements have implications for the understanding of intracellular biochemical networks.