Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli

Abstract

Protein mobility affects most cellular processes, such as the rates of enzymatic reactions, signal transduction, and assembly of macromolecular complexes. Despite such importance, little systematic information is available about protein diffusion inside bacterial cells. Here we combined fluorescence recovery after photobleaching with numerical modeling to analyze mobility of a set of fluorescent protein fusions in the bacterial cytoplasm, the plasma membrane, and in the nucleoid. Estimated diffusion coefficients of cytoplasmic and membrane proteins show steep dependence on the size and on the number of transmembrane helices, respectively. Protein diffusion in both compartments is thus apparently obstructed by a network of obstacles, creating the so-called molecular sieving effect. These obstructing networks themselves, however, appear to be dynamic and allow a slow and nearly size-independent movement of large proteins and complexes. The obtained dependencies of protein mobility on the molecular mass and the number of transmembrane helices can be used as a reference to predict diffusion rates of proteins in Escherichia coli. Mobility of DNA-binding proteins apparently mainly depends on their binding specificity, with FRAP recovery kinetics being slower for the highly specific TetR repressor than for the relatively nonspecific H-NS regulator.

Figure 1

Example of a typical FRAP measurement. (A and B) FRAP image sequence for a cell expressing the CFP-CheW-YFP fusion protein. After the prebleach image was acquired, the cell was bleached on the left pole by a high intensity laser beam, and the recovery of fluorescence was followed for 30 s until the fluorescence was evenly distributed throughout the cell. Sample images at indicated time points (A) and corresponding fluorescence intensity profiles (B) are shown, with lighter gray indicating later time points. Profile of the bleach image in panel B is shown by the dotted line. Gradual decrease in the overall fluorescence intensity through the course of experiment is due to bleaching during image acquisition. (C) Fluorescence recovery for CFP-CheW-YFP in three cells, indicated by different symbols. Relative fluorescence in the region of interest (ROI) was calculated as described in Materials and Methods.

Figure 2

Computational description of FRAP experiments. (A) Assumed bleach profile, with indicated lengths of the bleach zone (L_B) and the excess bleaching zone (L_E) and of the residual fluorescence intensity (B_d). See Materials and Methods for details and values of the parameters used in simulations. (B) Simulated concentration profile at 0.34 s for a protein of CFP-CheW-YFP size (solid line), overlaid with the sample profile of a CFP-CheW-YFP experiment at the same time point. (C) One of the experimental recovery curve from Fig. 1C (shaded dots), fitted with the model (solid line).

Figure 3

Dependence of estimated diffusion coefficients on the bleach zone length (A) and the cell length (B). Experiments in panels A and B were performed for Crr-YFP or CFP-CheW-YFP, respectively. Recovery curves were binned according to the length of the bleached zone or cell length, respectively, and the model was simultaneously fit to the entire pool of data for each bin as described in the text. Mean values for the bin are shown. Error bars depict 95% confidence interval (CI_95%) of the fitting procedure.

Figure 4

Dependence of cytoplasmic protein diffusion on molecular mass. (A) Representative recovery curves for a small fusion protein (Crr-YFP, 45 kDa; solid circles) and a large fusion protein (DnaK-YFP, 95.8 kDa; shaded diamonds). Error bars (the standard error of measurement) are smaller than the marker sizes. (B) The apparent diffusion coefficients, determined by our model, as a function of the molecular mass. The data are fitted by an empirical function D = α(MM)^-2 + D₀, with α = 4.3 × 10³μm² s⁻¹ kDa² and D₀ = 0.65 μm² s⁻¹ (solid line). Error bars depict 95% confidence interval of the fitting procedure.

Figure 5

Dependence of protein diffusion in the inner membrane on the size of the transmembrane domain. The apparent diffusion coefficient of the membrane-bound YFP fusion proteins is plotted against the number N of predicted membrane-spanning helices. Two or three different proteins of different molecular mass were measured for four and 12 transmembrane helices. The data are fitted by an empirical function D = α(N)^-3 + D₀, with α = 12 μm² s⁻¹ and D₀ = 0.019 μm² s⁻¹ (solid line). Error bars depict 95% confidence interval of the fitting procedure.

Figure 6

Exchange rates of DNA binding proteins. The recovery curves for YFP fusions to a specific (TetR-YFP; triangles) and a nonspecific (H-NS-YFP; circles) DNA binding factors. Solid and shaded symbols indicate the fluorescence intensity profile of the bleached and unbleached nucleoid or fluorescent focus, respectively. Error bars depict the standard error of measurement.