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Abstract Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use¹⁹F nuclear magnetic resonance spectroscopy to follow the reversible, two‐state unfolding thermodynamics of the N‐terminal Src homology 3 domain of theDrosophilasignal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein–crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG–protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG–protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered. 

Abstract The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)™Escherichia colicells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mMupon induction with 0.4 mMisopropyl β‐d‐thiogalactoside. We discuss the results in terms of what can and cannot be learned from in‐cell protein NMR studies inE. coli. 

Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer,Escherichia colicells andXenopus laevisoocytes. The complex is more stable in cells than in buffer and more stable in oocytes thanE. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded thanE. colicells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment.