Effects of Biomolecular Crowding on Protein Stability
Creators: name: Sarkar, Mohona
Publisher: name: University of North Carolina at Chapel Hill
Year: 2014

description: The intracellular milieu is complex, heterogeneous and crowded-- an environment vastly different from dilute, buffered solutions where most biophysical studies are performed. The cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This exclusion arises from the sum of two phenomena: steric repulsions and chemical interactions, often called hard and soft interactions, respectively. Most efforts to understand crowding have focused on steric repulsions. Globular protein stability is the difference in free energy between the compact, biologically functional native state and the ensemble of less compact, nonfunctional denatured state. The hard-core repulsive component of crowding stabilizes globular proteins because the decrease in available volume favors compact species. The effect of soft interactions can be stabilizing or destabilizing. Soft repulsive interactions reinforce the stabilizing influence of hard-core repulsions. However, the equilibrium is shifted towards the denatured state in systems dominated by attractive non-specific interactions, because unfolding exposes more reactive surface. In Chapter 1, I introduce the concepts of hard and soft interactions in more depth and discuss how they are expected to affect the equilibrium thermodynamic stability of globular proteins. In Chapter 2, I describe experiments that test these concepts by using Escherichia coli cell lysates as the crowding agents, chymotrypsin inhibitor 2 (CI2) as the test protein and NMR-detected amide-proton exchange to measure stability. The lysate destabilizes CI2, and the destabilization increases with increasing lysate concentration. This observation shows that the cytoplasm interacts favorably, but non-specifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. In fact, the effects of the lysate are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, non-specific interactions. In Chapter 3, I test the idea that the net charge on the crowding proteins affects stability. To accomplish this goal, I isolated the anionic proteins from the lysate and used them as the crowding agent. CI2 is an anion under the chosen conditions, and, therefore, I expected the net repulsive interactions between CI2 and the crowders to increase the stability of CI2. Instead, the refined lysate also resulted in destabilization. Thus, even the anionic proteins, which have the same net charge as CI2, significantly interact with CI2's backbone non-specifically to overcome the stabilizing effect of steric repulsion. My in vitro studies show that weak chemical interactions play key roles in the cytosol. It will even be more difficult to identify soft interactions in living cells where reductionist approaches are difficult or impossible to apply. Nevertheless, endeavors aimed at quantifying soft interactions are essential for producing a physiologically relevant understanding of biophysics. Once the details of soft interactions are known, it should be possible to tune them so as to obtain bespoke behavior in test tubes and in cells. In summary, despite their weak and non-specific behavior, biologists of all types need to keep these interactions in mind when designing experiments to correlate in vitro studies with in vivo behavior.