THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYME MECHANISMS

这个实验室致力于研究蛋白质的结构和 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。 研究的主要课题 是猪胃蛋白酶原，一种分子量为 39，630，其在pH 6和8.5之间稳定。 低于pH 6 胃蛋白酶原通过其前44个氨基酸的蛋白水解损失来激活自身 氨基酸，以产生具有酶活性的蛋白质，胃蛋白酶。 胃蛋白酶只有在pH值低于6时才稳定。 两种蛋白质都是未折叠的 通过暴露于高pH值、温度或浓度的 变性剂，如尿素。 在这样的解折叠之后，胃蛋白酶原可以 当回到自然状态时，它会重新折叠成正常的结构。 而胃蛋白酶不能。 我感兴趣的是其中的机制 重折叠反应和序列变化的影响 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， 与化学修饰和肽化学一起， 天然和未折叠物种的结构已经被 表征了 使用快速动力学技术，例如停止- 流动和T-跳跃，中间，部分折叠的形式， 在折叠反应中检测到，它们的结构已经被 部分确定和化学反应， 将它们与所研究的原生和内折叠形式分开。

This laboratory is ergaged 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 behavior 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 infolded forms investigated.