THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYME MECHANISMS

这个实验室从事蛋白质结构和蛋白质的研究。 一种蛋白质分子被合成为一种 随机卷曲，可折叠成特定的二级和三级 结构，无需任何外部帮助。的主要主题是 猪胃蛋白酶原是一种分子水平的单体蛋白 重量=39,630，在pH为6到8.5之间稳定。以下 PH 6胃蛋白酶原通过蛋白水解性丢失其第一个44激活自身 氨基酸，以产生一种具有酶活性的蛋白质，胃蛋白酶。 胃酶只有在pH低于6时才稳定。这两种蛋白质都是展开的 通过暴露在高pH、高温度或高浓度的 变性剂，如尿素。在这样的展开之后，胃蛋白酶原可以 当恢复到原生状态时，重新折叠到其正常结构， 而胃酶则不能。我对它的机制很感兴趣 复性反应及其对序列变化的影响 这两种蛋白质的行为。使用技术，例如 紫外光、圆二色和荧光光谱， 与化学修饰和多肽化学一起， 原生和未折叠物种的结构已经被 特色化的。使用快速运动技术，例如停止- 流动和T形跳跃、中间、部分折叠的形式 在折叠反应中检测到，它们的结构已经 部分确定，以及化学反应的性质 将它们与原生的和未折叠的形式分开 调查过了。

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 it's 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. I am interested 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 the native and unfolded forms investigated.