Zaccai G, Cendrin F, Haik Y, Borochov N, Eisenberg H
(C.N.R.S., U.R.A. 1333) Institut Laue Langevin, Grenoble, France.
J Mol Biol. 1989 Aug 5;208(3):491-500. doi: 10.1016/0022-2836(89)90512-3.
Malate dehydrogenase from the extreme halophile, Halobacterium marismortui, is stable only in highly concentrated solutions of certain salts. Previous work has established that its physiological environment is saturated in KCl; it remains soluble is saturated NaCl or KCl solutions; also it unfolds in solutions containing less than 2.5 M-NaCl or -KCl, salt concentrations which are still relatively high. New data show that the structure of this enzyme can be stabilized in a range of high concentrations of Mg2+ or other "salting-in" ions, also with exceptional protein-solvent interactions. "Salting-in" ions, contrary to stabilizing protein structure, usually favour unfolding. These, and most other results concerning the structure, stability and solvent interactions of the protein cannot be understood in terms of the usual effects of salts on protein structure. In this paper, a novel stabilization model is proposed for halophilic malate dehydrogenase that can account for all observations so far. The model results from experiments on the protein in salt solutions chosen for their different effects on protein stability (potassium phosphate, a strongly "salting-out" agent, and MgCl2, which is "salting-in"), and previously published data from NaCl and KCl solutions (mildly "salting-out"). Enzymic activity and stability measurements were combined with neutron scattering, ultracentrifugation and quasi-elastic light-scattering experiments. The analysis showed that the structure of the protein in solution as well as the dominant stabilization mechanisms were different in different salt solutions in which this enzyme is active. Thus, in molar concentrations of phosphate ions, stabilization and hydration are similar to those of non-halophilic soluble proteins, in which the hydrophobic effect dominates. In high concentrations of KCl, NaCl or MgCl2, on the other hand, solution particles are formed in which the protein dimer interacts with large numbers of salt and water molecules (the mass of solvent molecules involved depends on the nature of the salt but it is approximately equivalent to the protein mass). It is proposed that, under these conditions, the hydrophobicity of the protein core is too weak to stabilize the folded structure and the main stabilization mechanism is the formation of co-operative hydrate bonds between the protein and hydrated salt ions. Model predictions are in agreement with all experimental results, such as the different numbers of solvent molecules found in the solution particles formed with different salts, the loss of the exceptional solvent interactions concomitant with unfolding at non-physiological salt concentrations, and the different temperature denaturation curves observed for different salt solutions.(ABSTRACT TRUNCATED AT 400 WORDS)
来自极端嗜盐菌——死海嗜盐杆菌的苹果酸脱氢酶，仅在某些盐的高浓度溶液中才稳定。先前的研究已经证实，其生理环境中KCl饱和；它在饱和NaCl或KCl溶液中仍可溶；而且在含有低于2.5M-NaCl或-KCl的溶液中会展开，而这些盐浓度仍然相对较高。新数据表明，该酶的结构可以在一系列高浓度的Mg2+或其他“盐溶”离子中得到稳定，同时还具有特殊的蛋白质-溶剂相互作用。与稳定蛋白质结构相反，“盐溶”离子通常有利于蛋白质展开。这些以及其他大多数关于该蛋白质结构、稳定性和溶剂相互作用的结果，无法用盐对蛋白质结构的通常影响来解释。在本文中，针对嗜盐苹果酸脱氢酶提出了一种新颖的稳定化模型，该模型可以解释迄今为止的所有观察结果。该模型源于对蛋白质在盐溶液中的实验，这些盐溶液因其对蛋白质稳定性的不同影响而被选用（磷酸钾，一种强“盐析”剂，以及MgCl2，它是“盐溶”的），以及先前发表的来自NaCl和KCl溶液（轻度“盐析”）的数据。酶活性和稳定性测量与中子散射、超速离心和准弹性光散射实验相结合。分析表明，该酶在不同活性盐溶液中，溶液中蛋白质的结构以及主要的稳定化机制是不同的。因此，在磷酸盐离子的摩尔浓度下，稳定化和水合作用与非嗜盐可溶性蛋白质相似，其中疏水作用占主导。另一方面，在高浓度的KCl、NaCl或MgCl2中，会形成溶液颗粒，其中蛋白质二聚体与大量盐分子和水分子相互作用（所涉及的溶剂分子质量取决于盐的性质，但大致相当于蛋白质质量）。有人提出，在这些条件下，蛋白质核心的疏水性太弱，无法稳定折叠结构，主要的稳定化机制是蛋白质与水合盐离子之间形成协同水合键。模型预测与所有实验结果一致，例如在不同盐形成的溶液颗粒中发现的不同数量的溶剂分子、在非生理盐浓度下展开时伴随的特殊溶剂相互作用的丧失以及不同盐溶液中观察到的不同温度变性曲线。
