Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HIT from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HIT) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HIT were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/ Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol(-1) as compared with the wild-type protein. The removal of each -CH2- group burial decreased the stability by 5.1 kJ mol(-1) on average in the mutant proteins of Tk-RNase HIT examined. This is comparable with the value of 5.3 kJ mol(-1) obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HIT examined exhibited faster unfolding compared with the wildtype protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HIT. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.