Protein mobility in the cytoplasm of Escherichia coli

The rate of protein diffusion in bacterial cytoplasm may constrain a variet y of cellular functions and limit the rates of many biochemical reactions i n vivo. In this paper, we report noninvasive measurements of the apparent d iffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleach ing of GFP fluorescence and by photoactivation of a red-emitting fluorescen t 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 7 2-kDa fusion protein composed of GFP and a cytoplasmically localized maltos e 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 expres sion; second, the addition to GFP of a small tag consisting of six histidin e residues reduces D-a to 4.0 +/- 2.0 mu m(2)/s. Thus, a single effective c ytoplasmic viscosity cannot explain all values of D-a reported here. These measurements have implications for the understanding of intracellular bioch emical networks.