Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation

In proteins, empirical correlations have shown that changes in heat capacity (DeltaCp) scale linearly with the hydrophobic surface area buried upon folding. The influence of DeltaC(p) on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to DeltaCps of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a DeltaC(p)s of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated DeltaC(p) by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the DeltaC(p). We weigh the strengths and limitations of this method for determining DeltaC(p) values. Finally, we discuss the data in light of the physical origins of the DeltaC(p)s for RNA folding and consider their impact on biological function.