Foord R L, Leatherbarrow R J
Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, U.K.
Biochemistry. 1998 Mar 3;37(9):2969-78. doi: 10.1021/bi9712798.
Osmolytes are small organic solutes produced by the cells of all organisms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macromolecules and so conserve biological activity. They do not interact with the macromolecule directly but act by altering the solvent properties in the cellular environment, and so their presence indirectly modifies the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymotrypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to monitor the effect of this osmolyte on amide hydrogen exchange rates, which allows a probe at discrete points within the protein structure. Hydrogen exchange rates of specific backbone amide protons provide information about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the presence of a high concentration of glycine osmolyte has a profound stabilizing effect on the proteins studied, which is accompanied by a large reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significant changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, and a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in the exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to the observed exchange rates, and no simple generalization allows prediction of the extent of stabilization at any individual location. The results are discussed in light of the available structures of the proteins studied.
渗透溶质是所有生物体（除嗜盐菌外）的细胞在高应激情况下（如极端盐浓度、高温等）产生的小有机溶质，用于稳定其大分子从而保存生物活性。它们不直接与大分子相互作用，而是通过改变细胞环境中的溶剂性质来起作用，因此它们的存在间接改变了蛋白质的稳定性。在本文中，我们研究了一种模型渗透溶质甘氨酸对两种蛋白质——胰凝乳蛋白酶抑制剂2和马心细胞色素c的稳定作用。我们使用核磁共振来监测这种渗透溶质对酰胺氢交换速率的影响，这使得能够在蛋白质结构内的离散点进行探测。特定主链酰胺质子的氢交换速率提供了有关将这些酰胺暴露于溶剂并允许交换发生的局部结构波动的信息。我们发现，高浓度甘氨酸渗透溶质的存在对所研究的蛋白质具有深远的稳定作用，同时大多数缓慢交换的酰胺质子的交换速率常数大幅降低。光谱表明，这是在三维结构没有显著变化的情况下出现的。然而，对单个蛋白质内单个酰胺质子的影响并不均匀，观察到的影响幅度有很大差异。这一范围从交换速率没有明显变化到交换速率常数降低超过2个数量级。渗透溶质似乎改变了许多导致观察到的交换速率的不同过程，并且没有简单的概括能够预测任何单个位置的稳定程度。根据所研究蛋白质的现有结构对结果进行了讨论。
