The intracellular environment affects protein-protein interactions.

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 coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. 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 than E. coli cells, 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.

A Difference between In Vitro and In-Cell Protein Dimer Formation.

The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and Escherichia coli cells by using heteronuclear, multidimensional nuclear magnetic resonance spectroscopy. In buffer, the monomer is a partially folded molten globule, but that species is not observed in cells. Experiments using urea suggest that the monomer is unfolded in cells, but again, the molten-globule form of the monomer is absent. The data suggest that attractive chemical interactions in the cytoplasm unfold the molten globule. We conclude that the intracellular environment not only modulates the stability of protein complexes but also can change the species present, reinforcing the idea that chemical interactions are more important than hard repulsions in cells.

Protein shape modulates crowding effects.

Protein-protein interactions are usually studied in dilute buffered solutions with macromolecule concentrations of <10 g/L. In cells, however, the macromolecule concentration can exceed 300 g/L, resulting in nonspecific interactions between macromolecules. These interactions can be divided into hard-core steric repulsions and "soft" chemical interactions. Here, we test a hypothesis from scaled particle theory; the influence of hard-core repulsions on a protein dimer depends on its shape. We tested the idea using a side-by-side dumbbell-shaped dimer and a domain-swapped ellipsoidal dimer. Both dimers are variants of the B1 domain of protein G and differ by only three residues. The results from the relatively inert synthetic polymer crowding molecules, Ficoll and PEG, support the hypothesis, indicating that the domain-swapped dimer is stabilized by hard-core repulsions while the side-by-side dimer shows little to no stabilization. We also show that protein cosolutes, which interact primarily through nonspecific chemical interactions, have the same small effect on both dimers. Our results suggest that the shape of the protein dimer determines the influence of hard-core repulsions, providing cells with a mechanism for regulating protein-protein interactions.

Rheostatic Control of Protein Expression Using Tuner Cells.

We assessed the ability of two strains of Escherichia coli, BL21 (DE3) and Tuner (DE3), to express a variant of the B1 domain of protein G, which forms a side-by-side dimer, by using fluorine-labeling and 19F nuclear magnetic resonance spectroscopy. BL21 cells express the protein in a binary, all-or-none, manner, where more cells express the protein at a high level with an increasing inducer concentration. Tuner cells express the protein in a rheostatic manner, where expression increases across all cells with an increasing inducer concentration.

Surface Charge Modulates Protein-Protein Interactions in Physiologically Relevant Environments.

Protein-protein interactions are fundamental to biology yet are rarely studied under physiologically relevant conditions where the concentration of macromolecules can exceed 300 g/L. These high concentrations cause cosolute-complex contacts that are absent in dilute buffer. Understanding such interactions is important because they organize the cellular interior. We used 19F nuclear magnetic resonance, the dimer-forming A34F variant of the model protein GB1, and the cosolutes bovine serum albumin (BSA) and lysozyme to assess the effects of repulsive and attractive charge-charge dimer-cosolute interactions on dimer stability. The interactions were also manipulated via charge-change variants and by changing the pH. Charge-charge repulsions between BSA and GB1 stabilize the dimer, and the effects of lysozyme indicate a role for attractive interactions. The data show that chemical interactions can regulate the strength of protein-protein interactions under physiologically relevant crowded conditions and suggest a mechanism for tuning the equilibrium thermodynamics of protein-protein interactions in cells.

Macromolecular Crowding Is More than Hard-Core Repulsions.

Cells are crowded, but proteins are almost always studied in dilute aqueous buffer. We review the experimental evidence that crowding affects the equilibrium thermodynamics of protein stability and protein association and discuss the theories employed to explain these observations. In doing so, we highlight differences between synthetic polymers and biologically relevant crowders. Theories based on hard-core interactions predict only crowding-induced entropic stabilization. However, experiment-based efforts conducted under physiologically relevant conditions show that crowding can destabilize proteins and their complexes. Furthermore, quantification of the temperature dependence of crowding effects produced by both large and small cosolutes, including osmolytes, sugars, synthetic polymers, and proteins, reveals enthalpic effects that stabilize or destabilize proteins.Crowding-induced destabilization and the enthalpic component point to the role of chemical interactions between and among the macromolecules, cosolutes, and water. We conclude with suggestions for future studies.

Controlling and quantifying protein concentration in Escherichia coli.

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 coli cells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mM upon induction with 0.4 mM isopropyl ß-d-thiogalactoside. We discuss the results in terms of what can and cannot be learned from in-cell protein NMR studies in E. coli.