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Thirty percent of a cell's volume is filled with macromolecules, and protein chemistry in a crowded environment is predicted to differ from that in dilute solution. We quantified the effect of crowding by globular proteins on the equilibrium thermodynamic stability of a small globular protein. Theory has long predicted that crowding should stabilize proteins, and experiments using synthetic polymers as crowders show such stabilizing effects. We find that protein crowders can be mildly destabilizing. The destabilization arises from a competition between stabilizing excluded-volume effects and destabilizing nonspecific interactions, including electrostatic interactions. This competition results in tunable stability, which could impact our understanding of the spatial and temporal roles of proteins in living systems.

An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells.

The intracellular milieu is complex, heterogeneous and crowded-an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.

Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMRdetected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.M acromolecules in Escherichia coli reach concentrations of 300-400 g/L and occupy up to 40% of the cellular volume (1), but proteins are normally studied in buffer alone. The effects of crowding arise from two phenomena, hard-core repulsions and nonspecific chemical (soft) interactions (2-9). Hard-core repulsions limit the volume available to biological macromolecules for the simple reason that two molecules cannot be in the same place at the same time. This press for space favors compact states over expanded states. The second phenomenon arises because crowders not only exclude volume, but also participate in chemical interactions. Even though individually weak, the high concentration of macromolecules can lead to a large net effect. Repulsive nonspecific interactions reinforce the hardcore repulsions, whereas attractive nonspecific interactions oppose them. We use the term "nonspecific attractive interactions" to distinguish these from specific chemical interactions, such as ligand binding.Our aim is to understand how the crowded and heterogeneous, intracellular environment affects the equilibrium thermodynamic stability of globular proteins. Globular proteins are marginally stable in buffer at room temperature (10), with Gibbs free energy differences of 5-15 kcal/mol between the efficiently packed native (N) state and the ensemble of higher-energy denatured (D) states ðΔG o′ D Þ (11). Crowding effects arise from entropic and enthalpic contributions; hard-core repulsions are entropic, whereas the consequent nonspecific chemical interactions are also enthalpic. Hard-core repulsions always increase ΔG o′ D for globular proteins because D occupies more space than N (12-14). However,...