THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYME MECHANISMS

蛋白质结构和酶机制的热力学和动力学研究

• 批准号: 3854546
• 负责人: P MCPHIE
• 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/3854546
• 关键词: acidity /alkalinity; chemical stability; circular dichroism; enzyme mechanism; enzyme structure; fluorescence spectrometry; pepsin; pepsinogens; protein engineering; protein folding; protein sequence; protein structure function; stop flow technique; temperature jump; thermodynamics; ultraviolet spectrometry

This laboratory is engaged in studies on protein structure and the mechanism by which a protein molecule, which is synthesized as a random coil, can fold into a specific secondary and tertiary structure, without any external help. 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. Both proteins are unfolded by exposure to high pH, temperature or concentrations of denaturants, such as urea. After such unfolding, pepsinogen can refold to its normal structure, when returned to native conditions, whereas pepsin cannot. Interest is in the mechanism of this refolding reaction and on the influence of the change in sequence on the behaviour of the two proteins. Using techniques such as ultra-violet, circular dichroic and fluorescence spectroscopies, together with chemical modification and peptide chemistry, the structures of the native and unfolded species have been characterized. Using rapid kinetic techniques, such as stopped-flow and T-jump, intermediate, partly folded forms have been detected 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结构的研究。 一种蛋白质分子的合成机制， 无规卷曲，可以折叠成特定的二级和三级结构， 没有任何外部帮助。研究的主要对象是猪 胃蛋白酶原，分子量= 39，630的单体蛋白质， 在pH值6到8.5之间是稳定的pH低于6时胃蛋白酶原会自行激活 通过蛋白水解损失其前44个氨基酸，以产生 酶活性蛋白质胃蛋白酶。胃蛋白酶只有在pH值低于 6.这两种蛋白质都通过暴露于高pH、温度或pH值而解折叠。 浓度的变性剂，如尿素。在这样的展开之后， 当胃蛋白酶原恢复到天然状态时， 条件，而胃蛋白酶不能。兴趣在于这一机制 重折叠反应和序列的变化对 这两种蛋白质的行为。使用技术如 紫外、圆二色性和荧光光谱， 通过化学修饰和肽化学， 已经表征了天然和未折叠的物种。使用快速 动力学技术，如停流和T跳，中间， 在折叠反应中检测到部分折叠的形式， 结构已经部分确定， 化学反应将它们从天然的和未折叠的 调查的形式。