31] Calorimetric analyses of hyperthermophile proteins

Abstract

The enhanced stability of hyperthermophile proteins relative to those of mesophiles is the result of an interplay of many different forces. It would appear that nature uses all means at her disposal. In some thermophile proteins, there is an intrinsic increase in stability through increased numbers of electrostatic interactions and more efficient hydrophobic core packing. However, in some, the intrinsic stability is only marginal at the physiological growth temperature, and these are stabilized by ligand binding and modification of solvent structure through increased salt and osmolyte concentrations. A determination of the relative importance of any one of these factors requires a quantitative measure of its contribution. Calorimetry is often the easiest and most reliable method for determining free energy, enthalpy, and heat capacity changes accompanying biochemical reactions. The Gibbs free energy change quantitatively describes protein stability in the case of protein folding and ligand affinity in the case of association. Most importantly for thermophile studies, the state functions also provide the temperature dependence of these processes. The temperature dependence of the free energy is determined by the enthalpy change associated with the reaction through the van't Hoff relation. The enthalpy change is also usually temperature dependent as determined by the change in heat capacity. The chapter describes the practical use of differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) to measure protein stability and binding constants at high temperature.