Energetic evidence for formation of a pH-dependent hydrophobic cluster in the denatured state of Thermus thermophilus ribonuclease H.

NMR studies on the denatured states of proteins indicate that residual structure often resides predominantly in hydrophobic clusters. Such hydrophobic cluster formation implies burial of apolar surface and, consequently, is expected to cause a decrease in heat capacity. We report here that, in the case of ribonuclease H from the thermophile Thermus thermophilus, a sharp decrease in denatured-state heat capacity occurs at about pH 3.8; this result points to the formation of hydrophobic clusters triggered by the protonation of several (about four) carboxylic acid groups, and indicates that the burial of apolar surface is favored by the less hydrophilic character of the uncharged forms of Asp and Glu side-chains. The process is not accompanied by large changes in optically active structure, but appears to be highly cooperative, as indicated by the sharpness of the pH-induced transition in the heat capacity. This acid-induced hydrophobic burial in denatured T.thermophilus ribonuclease H is clearly reflected in the pH dependence of the denaturation temperature (i.e. an abrupt change of slope at about pH 3.8 is seen in the plot of denaturation temperature versus pH), supporting a role for such denatured-state hydrophobic clusters in protein stability. The finding of cooperative protonation of several groups coupled to surface burial in denatured T.thermophilus ribonuclease H emphasizes the potential complexity of denatured-state electrostatics and advises caution when attempting to predict denatured-state properties on the basis of simple electrostatic models. Finally, our results suggest a higher propensity for hydrophobic cluster formation in the denatured state of T.thermophilus ribonuclease H as compared with that of its mesophilic counterpart from Escherichia coli.