OSMOLYTE PROTECTION OF PROTEINS AGAINST INACTIVATION

渗透剂保护蛋白质免遭失活

• 批准号: 2629044
• 负责人: DAVID W BOLEN
• 金额: $ 16.5万
• 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 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2629044
• 关键词: alkaline phosphatase; arginine; betaine compound; bioenergetics; cell osmotic pressure; chemical stability; electrolytes; enzyme activity; gel filtration chromatography; glycerophosphorylcholine; inositol; nuclear magnetic resonance spectroscopy; pancreatic ribonuclease; proline; protein denaturation; protein structure function; solute; sorbitol; thermodynamics; thermostability; urea; valine

To adapt to harsh environmental stresses such as extremes of temperature, dehydration, and high osmotic pressure, certain organisms have accumulated intracellular concentrations of solutes called 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 intracellulary 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 our research are two fold: (I) to understand the mechanisms by which natural occurring osmolytes protect proteins against deleterious structural effects brought about by stresses of heat, cold, high salt, and urea normally encountered in living systems, and (II) to understand the mechanisms by which compatible and counteracting osmolytes ensure appropriate functional activity of proteins in the fact of these stresses. A major focus will be on osmolytes of mammalian kidney, including the compatible osmolytes sorbitol and inositol, and the counteracting osmolytes glycerolphosphoryl choline (GCP) and betaine. We will use transfer free energy measurements of amino acid side chains and the peptide backbone from water to proline, valine, arginine, sorbitol, inositol, GPC, and betaine, and to GPC/urea and sorbitol/urea mixtures. These data will be used to evaluate the transfer free energies of native and fully unfolded forms of RNase A from water to these solute-containing solutions. The objective is to determine which functional groups on the protein are responsible for protein stabilization, and in the case of arginine and valine as solutes, to determine which are responsible for protein destabilization. Such information is essential for understanding the molecular bases for protein stabilization by osmolytes. The effects of these solutes on NMR-detected hydrogen exchange (HX) kinetics of RNase A amide protons will be determined for the purpose of investigating the molecular basis of compatibility and counteraction. In the case of urea affects on protein structural integrity, HX will be used to determine how kidney osmolytes offset these effects.

适应苛刻的环境压力，例如极端 温度，脱水和高渗透压，某些生物 已经积聚了称为的溶质的细胞内浓度 渗透。 渗透压保护蛋白质和其他细胞成分 在极端条件下，并没有显着改变 大分子的生物学活性。 这些特征 在功能上将这些渗透压描述为兼容。 在改编中 哪个尿素集中在细胞中，如鲨鱼和哺乳动物 肾脏，甲胺渗透剂是浓缩的细胞内以保护的 细胞蛋白针对尿素的有害作用。 这些渗透压 据说是反对的，被认为会改变生物学 蛋白质在与尿素相反的方向上的活性。 长期 我们研究的术语目标是两个方面：（i）了解 自然发生的渗透剂保护蛋白质免受的机制 热，冷的压力带来的有害结构效应 高盐和尿素通常在生活系统中遇到，（ii） 了解兼容和抵消的机制 渗透液在事实上确保蛋白质的适当功能活性 这些压力。 重点将放在哺乳动物的渗透率上 肾脏，包括兼容的渗透虫山梨糖醇和肌醇，以及 抵消渗透液甘油磷酸胆碱（GCP）和 甜菜碱。 我们将使用氨基酸侧链的转移自由能测量 肽主链从水到脯氨酸，缬氨酸，精氨酸， 山梨糖醇，肌醇，GPC和BETAINE，以及GPC/尿素和山梨糖醇/尿素 混合物。 这些数据将用于评估传输的免费 RNase A的天然和完全展开形式的能量从水到水 这些含溶质的解决方案。 目的是确定哪个 蛋白质上的功能组负责蛋白质 稳定，在精氨酸和缬氨酸作为溶质的情况下， 确定哪些负责蛋白质不稳定。 这样的 信息对于理解分子碱基至关重要 渗透剂的蛋白质稳定。 这些溶质对 RNase A酰胺质子的NMR检测的氢交换（HX）动力学 将确定为了研究分子基础的目的 兼容性和反作用。 在尿素的情况下会影响 蛋白质结构完整性，HX将用于确定肾脏的方式 渗透压抵消了这些效果。