Abstract
Ribonucleases H from the thermophilic bacterium Thermus thermophilus and the mesophile Escherichia coli demonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (DeltaCp degrees ). The lower DeltaCp degrees of the thermophilic protein directly contributes to its higher thermal denaturation temperature (Tm). We propose that this DeltaCp degrees difference originates from residual structure in the unfolded state of the thermophilic protein; we verify this hypothesis by using a mutagenic approach. Residual structure in the unfolded state may provide a mechanism for balancing a high Tm with the optimal thermodynamic stability for a protein's function. Structure in the unfolded state is shown to differentially affect the thermodynamic profiles of thermophilic and mesophilic proteins.