Figure 5.

Testing for ensemble epistasis in the S100A4 protein. (A) Three-conformation ensemble of the S100A4 protein. The apo conformation (apo, slate, PDB: 1M31) is in equilibrium with the $C{a}^{2+}$ bound (ca, purple, PDB: 2Q91) and $C{a}^{2+}$/peptide bound (capep, green, PDB: 5LPU) conformations when $C{a}^{2+}$ (lime green spheres) and peptide (dark green) are present. (B) The relative populations of the apo, ca, and capep conformations change as $C{a}^{2+}$ concentration increases in the presence of saturating peptide. The magnitude of ensemble epistasis observed is $C{a}^{2+}$-dependent, because only some $C{a}^{2+}$ concentrations lead to multiple populated conformations. (C) Assigned energies ($\mathit{kcal}·mo{l}^{-1}$) of S100A4 conformations. Apo is most stable when peptide, μ_peptide, and $C{a}^{2+}$ chemical potentials, ${\mu }_{C{a}^{2+}}$, are zero (dashed lines). Capep is stabilized by increasing ${\mu }_{\mathit{peptide}}=20\hspace{0.17em}{\text{kcal}·\text{mol}}^{-1}$ (dark green arrow, solid green line). Increasing ${\mu }_{C{a}^{2+}}$ alters the energies of both ca and capep (lime green arrow, solid lines). All calculations were done at $T=298\hspace{0.17em}K$.