Thermodynamics Of Protein Denaturation At Temperatures Over 100 Degrees C: Cuta1 Mutant Proteins Substituted With Hydrophobic And Charged Residues

Although the thermodynamics of protein denaturation at temperatures over 100 degrees C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (T-d) ranging from 101 to 113 degrees C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (T-d = 137 degrees C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (Delta H) with no entropic gain from hydrophobic solvation around 100 degrees C, and that the stabilization due to salt bridges resulted from both the increase in Delta H from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 degrees C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.