A thermodynamic comparison of mesophilic and thermophilic ribonucleases H

The mechanisms by which thermophilic proteins attain their increased thermo stability remain unclear, as usually the sequence and structure of these pr oteins are very similar to those of their mesophilic homologues. To gain in sight into the basis of thermostability, we have determined protein stabili ty curves describing the temperature dependence of the free energy of unfol ding for two ribonucleases H, one from the mesophile Escherichia coli and o ne from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concen tration, and the resulting free energies of unfolding were fit to the Gibbs -Helmholtz equation to obtain a set of thermodynamic parameters for these p roteins. Although the maximal stabilities for these proteins occur at simil ar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In additi on, the stabilities of these proteins are similar at the optimal growth tem peratures for their respective organisms, suggesting that a balance of ther modynamic stability and flexibility is important for function.