Stability against Denaturation Mechanisms in Halophilic Malate Dehydrogenase "Adapt" to Solvent Conditions

Abstract

Malate dehydrogenase from Haloarcula marismortui (hMDH) is active, soluble and mildly unstable in an unusually wide range of salt conditions and temperatures, making it a particularly interesting model for the study of solvent effects on protein stability. Its denaturation (loss of activity due to concomitant dissociation and unfolding) kinetics was studied as a function of temperature and concentration of NaCl, potassium phosphate or ammonium sulphate in H 2O or 2H 2O. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when 2 H 2O replaced H 2O. Each salt condition was associated with a particular energy regime that dominated stability. In NaCl/H 2O, a positive enthalpy term, Δ H ≠O, always dominated the activation free energy of denaturation, Δ G ≠O. In potassium phosphate/H 2O and ammonium sulphate/H 2O, on the other hand, stability was dominated by a negative activation entropy, Δ S ≠O, and Δ H ≠O changed sign between 10°C and 20°C, consistent with a strong hydrophobic effect contribution, in these salting-out solvents. Decreasing stability at low temperatures, favouring cold denaturation, was observed. Replacing H 2O by 2 H 2O strengthened the hydrophobic effect in all conditions. As a consequence, conditions were found in which HMDH was not halophilic; below 10°C, it was stable in ≈0?1 M NaCl/ 2H 2O. The solution structure and preferential solvent interactions of hMDH in H 2O or 2H 2O solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H 2O or 2H 2O, an experimentally identical invariant solution particle was formed in both solvents. It had a total volume of 1.165 cm 3 g -1 and bound about 0.4 g of H 2O (0.44 g of 2H 2O) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.