Size Dependence of Protein Diffusion in the Cytoplasm of Escherichia coli

Abstract

Diffusion in the bacterial cytoplasm is regarded as the primary method of intracellular protein movement and must play a major role in controlling the rates of cell processes. A number of recent studies have used green fluorescent protein (GFP) tagging and fluorescence microscopy to probe the movement and distribution of proteins in the bacterial cytoplasm. However, the dynamic behavior of indigenous proteins must be controlled by a complex mixture of specific interactions, combined with the basic physical constraints imposed by the viscosity and macromolecular crowding of the cytoplasm. These factors are difficult to unravel in studies with indigenous proteins. To what extent the addition of a GFP tag might affect the movement of a protein through the cytoplasm has also remained unknown. To resolve these problems, we have carried out a systematic study of the size dependence of protein diffusion coefficients in the Escherichia coli cytoplasm, using engineered GFP multimers (from 2 to 6 covalently linked GFP molecules). Diffusion coefficients were measured using confocal fluorescence recovery after photobleaching (FRAP). At least up to 110 kDa (four linked GFP molecules), the diffusion coefficient varies with size roughly as would be predicted from the Einstein-Stokes equation for a classical (Newtonian) fluid. Thus, protein diffusion coefficients are predictable over this range. GFP tagging of proteins has little impact on the diffusion coefficient over this size range and therefore need not significantly perturb protein movement. Two indigenous E. coli proteins were used to show that their specific interactions within the cell are the main controllers of the diffusion rate.

Spatial Distribution and Diffusive Motion of RNA Polymerase in Live Escherichia coli

“…For these reasons, we speak of RNAP diffusion and the RNAP spatial distribution even though we are actually measuring the fluorescence from ␤Ј-EGFP. In addition, because we are tagging the large RNAP by a single copy of the small EGFP, we expect the dynamics measured to be indicative of the true RNAP dynamics (24).…”

Protein Interactions in the Escherichia coli Cytosol: An Impediment to In‐Cell NMR Spectroscopy

“…[1] With the growing awareness of this deficiency there is a drive towards studying proteins under physiologically relevant conditions, [3][4][5][6][7][8] including the application of spectroscopic techniques to proteins inside cells. [9][10][11] In-cell NMR spectroscopy provides the means to monitor protein conformation and dynamics at atomic resolution inside living cells. [11-24] 15 N-labelled "biologically inert" proteins overexpressed in the host E. coli, yield in-cell spectra closely similar to the spectrum of the purified protein.…”

The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

“…They collected literature data [12,13,[65][66][67][68][69][70][71][72][73][74][75][76][77] of diffusion coefficients in the cytoplasm of E. coli and used Equation 5 to fit the data. From this fit they determined ξ of around 0.5 nm and R h ≈ 42 nm [15].…”