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.

Proteins rarely work alone. Networks of protein–protein interactions turn a myriad of chemical signals into physiological responses that maintain intracellular homeostasis (1). Therefore, it is unsurprising that nearly two-thirds of disease-causing missense mutations involve protein complexes (2). However, nearly all equilibrium thermodynamic and kinetic studies of protein–protein interactions have been performed in dilute buffer. Acquiring quantitative equilibrium data on protein complexes in cells, despite the fact that living things are not at equilibrium, is important for two reasons. First, the equilibrium condition is the starting point for estimating the driving force under nonequilibrium conditions (3). Second, recent results on protein stability show that conclusions from experiments conducted in dilute solution cannot be extrapolated to the crowded and complex intracellular environment (4).The cellular interior contains a variety of proteins, nucleic acids, and small molecules. In the bacterium Escherichia coli, the concentration of macromolecules can exceed 300 g/L and occupy up to 30% of volume (5). The macromolecule concentration in eukaryotic cells is smaller (6) but still hundreds of times larger than the solute concentrations used in most in vitro studies. Furthermore, the majority of proteins in the cells studied here, E. coli and oocytes, are net polyanions with mean isoelectric points of 6.6 and 6.7, respectively (7). Understanding how the intracellular environment modulates protein—and protein complex—stability is important because the totality of weak interactions in cells form the so-called quinary structure that organizes the crowded cellular interior (8–10). Importantly, these interactions cannot be measured in dilute solutions.The physical consequences of macromolecular crowding arise from two components: hard-core repulsions and so-called “soft” chemical interactions (11). Hard-core repulsion is a steric effect, arising from the impenetrable nature of atoms. Steric repulsions reduce the volume available to reactants and products. Simple theory predicts that sterics favor compact states (12). For small, folded proteins, stability is described by the two-state equilibrium between the biologically active folded state and the inactive, higher-energy, and less-compact unfolded state (13). For protein complex formation involving folded proteins, which comprises an equilibrium between constituent proteins and the complex, the complex exposes less surface than the sum of the two monomers (14). Thus, simple theory predicts stabilization of proteins and protein complexes, although more sophisticated theoretical efforts focused on the size of crowding molecule predict stabilization or destabilization (15, 16).Soft interactions include hydrogen bonds and charge–charge, water–protein, solute–protein, and hydrophobic interactions. Of these, the only strong repulsive interaction is that from the opposition of like charges. These repulsions add to the hard-core effect and are therefore stabilizing. The other soft interactions are attractive and destabilizing because they favor expanded conformations that allow access to the attractive surfaces.Until recently the focus was on hard-core interactions and crowding (17) because studies of protein stability (quantified as the free energy of unfolding, ΔGUo'') in uncharged synthetic polymers tended to show only a stabilizing influence compared to buffer alone (11). However, recent studies of stability under more physiologically relevant crowded conditions and in living cells show, by and large, a decrease in stability (4). Studies of protein–protein interactions in vitro under physiologically relevant crowded conditions also show the importance of chemical interactions (18, 19).Yeast two-hybrid techniques, coimmunoprecipitation, and split systems provide simple yes/no information about protein interactions in cells, however, these methodologies are also prone to false positives. The protein–protein interface can also be characterized by in-cell NMR. However, quantification of the equilibrium thermodynamics of complex stability is challenging in cells. The few reports tend to involve heterodimerization (20), which can be complicated to assess because the concentration of both reactants must be controlled, fluorescence-resonance-energy transfer because labels adding hundreds to thousands of daltons must be included, or because competition with natural, unlabeled proteins in cells must be considered (21–27).