Crowding and confinement can oppositely affect protein stability.

Proteins encounter crowded and confined macromolecular milieus in living cells. Simple theory predicts that both environments entropically stabilize proteins if only hard-core repulsive interactions are considered. Recent studies show that chemical interactions between the surroundings and the test protein also play key roles such that the overall effect of crowding or confinement is a balance of hard-core repulsions and chemical interactions. There are, however, few quantitative studies. Here, we quantify the effects of crowding and confinement on the equilibrium unfolding-thermodynamics of a model globular protein, KH1. The results do not agree with predictions from simple theory. KH1 is stabilized by synthetic-polymer crowding agents, but destabilized by confinement in reverse micelles. KH1 is more entropically stabilized and enthalpically destabilized in concentrated solutions of the monomers than it is in solutions of the corresponding polymer. When KH1 is confined in reverse micelles, the temperature of maximum stability decreases, the melting temperature decreases, and the protein is entropically destabilized and enthalpically stabilized. Our results show the importance of chemical interactions to protein folding thermodynamics and imply that cells utilize chemical interactions to tune protein stability.