Bonneté F, Madern D, Zaccaï G
Institut Laue Langevin, Grenoble, France.
J Mol Biol. 1994 Dec 9;244(4):436-47. doi: 10.1006/jmbi.1994.1741.
Malate dehydrogenase from Haloarcula marisomortui (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 2H2O. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when 2H2O 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 to 0, always dominated the activation free energy of denaturation, delta G not equal to 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 to 0. and delta H not equal to 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 2H2O 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 approximately 0.1 M NaCl/2H2O. The solution structure and preferential solvent interactions of hMDH in H2O or 2H2O solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H2O or 2H2O, an experimentally identical invariant solution particle was formed in both solvents. It had a total volume of 1.165 cm3 g-1 and bound about 0.4 g of H2O (0.44 g of 2H2O) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.
来自死海嗜盐菌（Haloarcula marisomortui）的苹果酸脱氢酶（hMDH）在异常宽泛的盐浓度条件和温度范围内具有活性、可溶且稳定性稍差，这使其成为研究溶剂对蛋白质稳定性影响的一个特别有趣的模型。研究了其变性（由于伴随解离和去折叠而导致活性丧失）动力学与温度以及H₂O或²H₂O中NaCl、磷酸钾或硫酸铵浓度的函数关系。对数据应用了过渡态理论分析。在所有情况下，稳定性（抗变性能力）都随盐浓度的增加以及²H₂O替代H₂O而增强。每种盐条件都与一种主导稳定性的特定能量状态相关。在NaCl/H₂O体系中，一个正的焓项，即ΔH≠0，总是主导变性的活化自由能ΔG≠0。另一方面，在磷酸钾/H₂O和硫酸铵/H₂O体系中，稳定性由负的活化熵主导，即ΔS≠0，且在10℃至20℃之间ΔH≠0改变符号，这与在这些盐析溶剂中强烈的疏水效应贡献一致。观察到在低温下稳定性降低，有利于冷变性。在所有条件下，用²H₂O替代H₂O都增强了疏水效应。结果发现，在某些条件下hMDH并非嗜盐的；在低于10℃时，它在约0.1M NaCl/²H₂O中是稳定的。通过密度测定法和中子散射研究了hMDH在含NaCl的H₂O或²H₂O溶剂中的溶液结构和优先溶剂相互作用。尽管蛋白质在H₂O或²H₂O中的稳定性不同，但在两种溶剂中都形成了实验上相同的不变溶液颗粒。它的总体积为1.165 cm³ g⁻¹，结合约0.4g的H₂O（0.44g的²H₂O）和约0.08g NaCl/g蛋白质。讨论了这些结果对涉及离子结合的hMDH稳定化模型的影响。
