Crowding and Confinement Effects on Protein Diffusion In Vivo

Abstract

Most biochemical and biophysical studies of the reaction mechanisms, kinetics, and thermodynamics of proteins and nucleic acids are carried out in vitro using dilute aqueous solutions of purified biopolymer constituents. However, the cytoplasm of both prokaryotic and eukaryotic cells contains a very high total concentration of proteins, nucleic acids, lipids, and supramolecular assemblies of these constituents. In Escherichia coli grown at moderate osmolality, the typical total mass density of protein and nucleic acid within the cytoplasm is ϳ220 mg/ml (5), distributed among the nucleoid, ribosomes, and a diverse collection of smaller proteins, tRNA, and mRNA (3). Taken together, these macromolecules occupy some 20 to 30% of the total cytoplasmic volume.In a crowded fluid, each molecule is excluded from much of the total volume by the presence of other biopolymers (11, 37). In thermodynamic terms, excluded volume decreases the translational entropy of each species, increases free energy, and, thus, increases the thermodynamic driving force to react or bind. These effects could be very large; the thermodynamic activity (the "effective concentration") of a typical globular protein could be Ͼ100 times higher in the E. coli cytoplasm than at the same concentration in an uncrowded solution (9). Crowding also dramatically affects diffusion (26), which is critical for normal cell function and growth (28). Excluded volume slows diffusion by making it less likely that a probe particle can find space in which to move without simultaneous, cooperative motion of several or many background particles (26).While the bacterial cytoplasm is often assumed to be a crowded aqueous solution (37), the physical state of the cytoplasm is uncertain. Particularly for the low water content induced by hyperosmotic stress, the cytoplasm might become a biopolymer meshwork comprising the nucleoid, associated proteins, nascent mRNA, ribosomes, polypeptide chains, and strongly associated water (13, 38). Such conditions are reminiscent of a polymeric hydrogel (1). Confinement within the pores of the meshwork would enhance protein binding equilibria and slow protein diffusion in a manner qualitatively similar to crowding.Strong effects of crowding and confinement in vitro have been observed for the tracer diffusion of globular proteins (26) and for the diffusion of tracer proteins in concentrated solutions of hydrophilic polymers (2, 8) and in hydrogels (1). The apparent diffusion coefficient decreases roughly exponentially with the macromolecular volume fraction , in agreement with a parametrized model called scaled particle theory (SPT) (14,26). Additional studies in vitro have shown crowding effects on protein folding (36) and association (24), on the thermodynamics of the protein-nucleic acid interactions critical to replication (27), on enzyme kinetics (25), and on the stability of protein oligomers such as F-actin (16, 20) and of fibrils such as ␤-amyloid (15).We know of no experimental studies of crowding/confinement effects on protein di...

Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence

“…5D) consistent with an earlier suggestion (41). It is interesting to note that the occurrence of similar empty spaces in the cell was reported as a result of osmotic upshift (53). However, it is not obvious that the molecular reasons for such a phenotype are same in these two cases.…”

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding

“…Molecular crowding has received little attention in living cells, although it is usually invoked to explain why biochemical reactions rates may vary in vivo and in vitro (26)(27)(28)(29). For instance, the diffusion coefficient of the green fluorescent protein is typically 10 times lower in cells than in vitro (30).…”

Macroscopic Transport Equations in Many-Body Systems from Microscopic Exclusion Processes in Disordered Media: A Review

“…Anomalous diffusion refers to phenomena that lead to a nonlinear growth of particles' mean square displacement. Deviation from the standard linear behavior arise, for example, when obstacles impede the motion of particles [17,21,22] or when distinguishable species compete for the available spatial resources [10,[23][24][25][26][27]. These conditions are certainly met when studying the molecular mobility inside a cell [28,29].…”

“…In this respect, living cells behave much like fractal or otherwise disordered systems [30,31]. However, strong evidences also exist in favor of normal (Brownian) diffusion, crowding, and confinement resulting in this scenario in (often nontrivial) modifications of the diffusion coefficient [10,27,32,33]. Moreover, as shown in Galanti et al [34], the effect of crowding can result in crossovers between normal and anomalous diffusion, leading to different descriptive scenarios which appear to depend on the selected initial conditions and on the specific time scale of observation.…”