THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYMIC MECHANISMS

蛋白质结构和酶机制的热力学和动力学研究

• 批准号: 3754086
• 负责人: P MC PHIE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3754086
• 关键词: chemical kinetics; circular dichroism; enzyme mechanism; fluorescence spectrometry; pepsin; pepsinogens; protein folding; protein sequence; protein structure; stop flow technique; temperature jump; thermodynamics; ultraviolet spectrometry

This laboratory is engaged in studies on protein structure and the mechanism of protein folding. The main subject of research is swine pepsinogen, a monomeric protein of molecular weight= 39,630, which is stable at pH's between 6 and 8.5. Below pH 6 pepsinogen activates itself by proteolytic loss of its first 44 amino acids, to produce an enzymatically active protein, pepsin. Pepsin is stable only at pH's below 6. Pepsin and pepsinogen are unfolded by exposure to high pH, temperature or concentrations of denaturants, such as urea. However, unfolded pepsinogen can refold to its normal structure, when returned to native conditions, whereas pepsin cannot. I am interested in the mechanism of this refolding reaction and how the difference in sequence influences the refolding of the two proteins. We have used techniques such as ultra-violet, circular dichroic and fluorescence spectroscopies, together with chemical modification and peptide chemistry, to characterize the structures of the native and unfolded species. We have used rapid kinetic techniques, such as stopped-flow and T-jump, to detect partly folded forms in the folding reaction; their structures have been partially determined and the nature of the chemical reactions which separate them from the native and unfolded forms investigated.

这个实验室从事蛋白质结构和蛋白质的研究。 蛋白质折叠的机制。研究的主要对象是猪。 胃蛋白酶原，是一种相对分子质量为39,630的单体蛋白质， 在pH值在6到8.5之间稳定。在pH 6以下，胃蛋白酶原自我激活 通过蛋白质分解损失其最初的44个氨基酸，产生一个 具有酶活性的蛋白质，胃酶。胃酶只有在pH值以下才稳定 以下6.胃酶和胃蛋白酶原通过暴露在高pH值下展开， 温度或变性剂的浓度，如尿素。然而， 未折叠的胃蛋白酶原可以重新折叠到其正常结构，当返回 原生条件，而胃酶不能。我对这个项目很感兴趣 这种复性反应的机制以及序列上的差异 影响这两种蛋白质的重折叠。我们使用了一些技术 例如紫外线、圆二色和荧光光谱， 再加上化学修饰和多肽化学， 描述原生物种和未展开物种的结构。我们有 使用快速动力学技术，如停流和T跳跃，以检测 在折叠反应中部分折叠的形式；它们的结构已经 以及部分确定的化学反应的性质 将它们与所调查的原生和展开的形式分开。