Effect of Molecular Crowding in Living Cells

The biochemical and biophysical principles discussed in preceding sections were derived mainly from in vitro studies using pure reactants, often small molecules, or in relatively low concentrations, for example, less than 1 mg ml^-1 of total macromolecule such as protein, nucleic acids, or polysaccharides. However, all cells contain various biomacromolecules at high concentration (Hoppert and Mayer, 1999).

Biochemical reactions in living cells occur in media crowded with other soluble or structured macromolecules, resulting in nonspecific interactions between individual macromolecules and their immediate surroundings in the cytosol.

These background interactions lead to three different phenomena: (1) macromolecular crowding, attributed to volume excluded by one soluble macromolecule to another; (2) macromolecular confinement, attributed to steric-repulsive interactions between the macromolecule of interest and its static boundaries; and (3) macromolecular adsorption, due to reversible association of a macromolecule to the surface of a nearby fiber or membrane (Minton, 2006).

Nonspecific interactions may either be repulsive, leading to preferential size and shape dependent exclusion, or attractive, leading to nonspecific binding or adsorption. Predominantly repulsive background interactions tend to enhance the rate and extent of macromolecular association in solution, while predominantly attractive background interactions tend to enhance the tendency of macromolecules to cluster nonspecifically or adsorb onto surfaces.

However, in a complex and heterogeneous medium of the cytoplasm, it is a challenge to discern whether the locally dominant background interactions are likely to be attractive or repulsive and to identify their effects on any specific reaction.

Molecular crowding, in principle, can markedly slow down the diffusion rate. Consequently crowding plays a role in all biological processes mediated by noncovalent associations or conformational changes of the macromocules, such as those involved in the synthesis of nucleic acids and proteins, intermediary metabolism and cell signaling, as well as the functioning of dynamic motile systems. In general, macromolecular crowding nonspecifically enhances reactions leading to the reduction of total excluded volume, independent of hydrogen bondings, van der Waals forces or charges.

These reactions include the formation of macromolecular complexes in the medium, binding of macromolecules to surface binding sites, formation of insoluble aggregates, as well as compaction or folding of proteins. Simple statistical-thermodynamic modeling studies reveal that the 'passive crowding macromolecules' could exert order-of-magnitude or greater changes in both the rates and equillibria of numerous reactions tested. To this end, one should also recognize that system studies via simulation are still models instead of the real thing.

Not all idiosyncratic details of the model system are of general value for understanding the real cellular system. Biological systems are more complex than theoretical or in vitro experimental studies because of enhanced heterogeneity and the presence of nonspecific repulsive and attractive intermolecular interactions in addition to volume exclusion.

Model studies also show that the magnitude of the effects is strongly dependent on the relative sizes and shapes of the concentrated crowding species used and on the nature of the macromolecular reactants and products.

However, to date, the results obtained via model simulation studies have provided important new insights for understanding the subject. In view of the complexity and heterogeneity of the intracellular fluids, results from simplified model studies can only partially address the complex problems encountered with the in vivo system (Zhou et al., 2008; Ellis, 2001).

The densely packed environment in the cytosol appears to impede the folding of relatively large polypeptides since their diffusion rates would be more drastically reduced relative to those of smaller polypeptides.

Furthermore, the presence of a large number of crowding macromolecules would increase the probability for a newly synthesized polypeptide to interact with other macromolecules before it can properly fold. To overcome these problems, nature makes use of a class of molecular chaperones as well as a number of protein disulfide isomerases to facilitate proper folding of nascent proteins, including those mediated by cysteine disulfide bond formation, to yield functional proteins.

As a result, proteins are found to be folded very efficiently when synthesized inside the cell. Anfinsen showed that it took several hours for ribonuclease-A to fold in the test tube, a rate much slower than the rate at which functional ribonuclease-A is produced in cells (about 2 min.).

A similar rationale was adopted in cell signaling. To facilitate cell signaling processes inside crowded cytosols, scaffold or anchorage proteins are adopted to generate signalsomes to process cell signaling. To this end, formation of intracellular Dishevelled-based signalsomes has been demonstrated to occur during the activation of Wnt signaling (Yokoyama et al., 2010).