| Abstract: | Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔHobs, and accounts for as much as a half of the observed heat capacity change, ΔCp. However, there is still a substantial ΔCp associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)34 to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)34 binding over this entire pH range, with contributions from at least three sets of protonation sites (pKa1 = 5.9–6.6, pKa2 = 8.2–8.4, and pKa3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔCp for the SSB‐dT(pT)34 interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol−1 K−1) accounts for as much as half of the total ΔCp. The values of the “intrinsic” ΔCp,0 range from −210 ± 33 cal mol−1 °K−1 to −237 ± 36 cal mol−1K−1, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔCp, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc. |
