Enthalpic and entropic consequences of the removal of disulfide bridges in ribonuclease A

In this study a careful analysis of the enthalpic and entropic effects associated with the removal of each of the four disulfide bridges in ribonuclease A is accomplished. The fundamental role of disulfides for the stability of the native structure is emphasized by the strong decrease in denaturation temperature, at least 20°C. In contrast to the traditional belief, the destabilization in two out of four mutant forms is driven by enthalpic factors, related to the loss or weakening of several van der Waals interactions among side-chains in the protein close-packed interior. Theoretical relations, derived in the assumption that the insertion of a disulfide does affect only the entropy of the denatured state by lowering its conformational freedom, prove to be unable to reproduce and explain the experimental results. Such failure is rationalized by taking into account the complexity of the protein structure in both the native and denatured states, and the large variety of stabilizing and destabilizing interactions involved.