STABILITY AGAINST DENATURATION MECHANISMS IN HALOPHILIC MALATE-DEHYDROGENASE ADAPT TO SOLVENT CONDITIONS

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 H2O or (H2O)-H-2. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when (H2O)-H-2 replaced H2O. Each salt condition was associated with a particular energy regime that dominated stability. In NaCl/H2O, a positive enthalpy term, Delta H-not equal 0 , always dominated the activation free energy of denaturation, Delta G(not equal 0) . In potassium phosphate/H2O and ammonium sulphate/H2O, on the other hand, stability was dominated by a negative activation entropy Delta S-not equal 0, and Delta H-not equal 0 changed sign between 10 degrees C and 20 degrees C, consistent With a strong hydrophobic effect contribution, in these salting-out solvents. Decreasing stability at low temperatures, favouring cold denaturation, was observed. Replacing H2O by (H2O)-H-2 strengthened the hydrophobic effect in all conditions. As a consequence, conditions were found in which hMDH was not halophilic; below 10 degrees C, it was stable in approximate to 0.1 M NaCl/(H2O)-H-2. The solution structure and preferential solvent interactions of hMDH in H2O or (H2O)-H-2 solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H2O or (H2O)-H-2, 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 H2O (0.44 g of (H2O)-H-2) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.