Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (ΔH_obs) and the negative heat capacity change (ΔC_p,obs) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined ΔH_obs over a much wider temperature range (5-60 °C) and as a function of monovalent salt concentration and type for SSB binding to (dT)₇₀ under solution conditions that favor the fully wrapped (SSB)₆₅ complex (monovalent salt concentration ≥0.20 M). Over this wider temperature range we observe a strongly temperature-dependent ΔC_p,obs. The ΔH_obs decreases as temperature increases from 5 to 35 °C (ΔC_p,obs <0) but then increases at higher temperatures up to 60 °C (ΔC_p,obs >0). Both salt concentration and anion type have large effects on ΔH_obs and ΔC_p,obs. These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 °C), the midpoint of which shifts to higher temperature (above 35 °C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br^- > Cl ^- > F^-, and these anions are then released upon binding ssDNA, affecting both ΔH_obs and ΔC_p,obs. We conclude that the experimentally measured values of ΔC_p,obs for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.