OSMOLYTE PROTECTION OF PROTEIN AGAINST INACTIVATION

渗透剂保护蛋白质免遭失活

• 批准号: 2187293
• 负责人: DAVID W BOLEN
• 金额: $ 14.57万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187293
• 关键词: alkaline phosphatase; betaine compound; biophysics; carboxypeptidase; chemical group; chemical kinetics; chemical stability; enzyme activity; hexokinase; hydropathy; intermolecular interaction; lactate dehydrogenases; nuclear magnetic resonance spectroscopy; osmotic pressure; pancreatic ribonuclease; proline; protein denaturation; protein structure function; sarcosine; solubility; solute; solutions; sucrose; thermodynamics; thermostability; urea; water solution

Heat, freezing, dehydration, and high osmotic pressure present serious stresses to a large number of plants and animals in the biosphere. In the study of the biology of adaptation it is found that organisms adapted to extreme environments accumulate significant intracellular concentrations of solutes termed 'osmolytes'. Osmolytes protect proteins and other cell components against the extreme conditions and do so without significantly altering the biological activity of the macromolecule. These characteristics functionally describe these osmolytes as 'compatible'. In adaptations in which urea is concentrated in cells, as in sharks and in mammalian kidney, methylamine osmolytes are concentrated intracellularly to protect cell proteins against the deleterious effects of urea. These osmolytes are said to be 'counteracting' and are believed to alter the biological activity of proteins in a direction opposite to that of urea. The long term goals of the proposed research are two fold: (I) to understand the mechanisms by which natural occurring osmolytes protect proteins against thermal inactivation, inactivation by denaturing solvents, and inactivation brought about by drying, and (II) to explore how osmolytes greatly affect protein stability without causing much of an effect on biological activity and protein structure. To understand the origin of the protecting effects of osmolytes we have begun determining transfer free energies of amino acids (deltaGtr) from water to osmolyte solutions. We have found that the solvophobic character of amino acids are qualitatively different in osmolyte solutions than they are in water, with the aromatic side chains favoring transfer to osmolyte and the aliphatic side chains opposing it. All side chain deltaGtr values are, however, small in magnitude and appear to contribute little to stabilization of the native state of the protein in osmolyte solutions. The most striking result of these studies so far is that the unfavorable transfer of the polypeptide backbone from water to osmolyte is the dominant factor preventing unfolding and therefore contributing to protein stability. It is clear that different principles appear to be operating when protein unfolding is being carried out in the presence of osmolytes as compared to water. The specific aims of this work is to investigate the-fundamental properties of side chains and peptide backbones in these solvent systems, to determine the effects of osmolyte concentration on enzyme kinetic parameters, and to evaluate the flexibility and internal dynamics of proteins in osmolytes by amide exchange kinetics using 2D NMR.

加热，冷冻，脱水和高渗透压 生物圈中大量动植物的压力。 在 对适应生物学的研究发现，有机体适应了 在极端环境中积累明显的细胞内 称为“渗透剂”的溶质浓度。 渗透剂保护蛋白质 和其他针对极端条件的细胞成分，这样做 没有显着改变 高分子。 这些特征在功能上描述了这些 Osmolytes为“兼容”。 在尿素浓缩的改编中 在细胞中，如鲨鱼和哺乳动物肾脏一样，甲胺渗透剂是 细胞内浓缩以保护细胞蛋白免受 尿素的有害作用。 据说这些渗透压是 “抵抗”，被认为会改变 蛋白质朝与尿素相反的方向。 长期目标 拟议的研究中有两个方面：（i）了解机制 自然发生的渗透剂可保护蛋白质免受热的影响 失活，通过贬值和失活而失活 通过干燥而带来的，（ii）探索渗透率如何极大地影响 蛋白质稳定性不会对生物产生很大影响 活性和蛋白质结构。 要了解我们拥有的渗透剂的保护作用的起源 开始确定氨基酸（Deltagtr）的自由能从 水到渗透溶液。 我们发现溶无精子 氨基酸的特征在渗透质中质量不同 解决方案比在水中的解决方案，芳香的侧链有利于 转移到Osmolyte和与之相对的脂肪族侧链。 全方位 然而，链deltagtr值的幅度很小，似乎 对蛋白质的天然状态的稳定很少 渗透液解决方案。 到目前为止，这些研究最引人注目的结果是 多肽主链从水到 渗透压是预防展开的主要因素，因此 有助于蛋白质稳定性。 显然，不同的原则 在蛋白质展开时似乎正在运行 与水相比的存在。 这个特定的目的 工作是研究侧链的基本特性， 这些溶剂系统中的肽骨架，以确定 在酶动力学参数上渗透浓度，并评估 酰胺中渗透剂中蛋白质的灵活性和内部动力学 使用2D NMR交换动力学。