The intracellular environment affects protein-protein interactions

Abstract

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.

Keywords: macromolecular crowding; protein; protein–protein interactions; thermodynamics.

Fig. 1.

Complex formation in E. coli. (A) Dissociation of the A34F GB1 side-by-side homodimer (Protein Data Bank [PDB] ID code 2RMM) showing the tryptophan at position 43. (B) ¹⁹F NMR spectra acquired at 298 K 6-fluorotryptophan–labeled protein in dilute solution 0.50 mM GB1 in 20 mM phosphate buffer (pH 7.5) and E. coli using inducer concentrations of 1.0 mM (green) and 0.24 mM (purple), with the cytosol buffered at pH 7.6. (C) Dissociation constants were quantified from the binding isotherms acquired in cells (green) and buffer (orange). Error bars represent the standard deviation of the mean from triplicate analysis.

Fig. 2.

Complex formation in X. laevis oocytes. (A) Dissociation of the A34F GB1 side-by-side homodimer (PDB ID code 2RMM) showing the tyrosines at positions 3, 33, and 45. (B) ¹⁹F NMR spectra acquired at 288 K of the 3-fluorotyrosine–labeled protein in 20 mM phosphate buffer (pH 7.4) and oocytes (black, as acquired; purple, deconvoluted dimer; green, deconvoluted monomer; and orange, sum of deconvolutions). (C) Dissociation constants were quantified from binding isotherms acquired in oocytes (green) and buffer (orange). The uncertainties, which are from least squares fitting, are smaller than the points.

Fig. 3.

Charge and dimer stability $\left(\mathrm{\Delta \Delta }{G}_{D\to M}^{o\text{'}}=\mathrm{\Delta }{G}_{D\to M,variant,buffer/cells}^{o^{\text{'}}}-\mathrm{\Delta }{G}_{D\to M,A34F,buffer}^{o\text{'}}\right)$. Positive values indicate increased stability and vice versa. Uncertainties are propagated from the uncertainties in $\mathrm{\Delta }{G}_{D\to M}^{o\text{'}}$. The absence of error bars indicates minimum magnitude of $\mathrm{\Delta \Delta }{G}_{D\to M}^{o\text{'}}$for variants exhibiting only dimer or monomer in cells.