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。 的主要课题 研究是猪胃蛋白酶原，一种分子的单体蛋白质， 重量= 39，630，其在pH 6和8.5之间稳定。 下面 pH 6胃蛋白酶原通过蛋白水解失去其前44 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、温度或高浓度的 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， 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 关于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，和化学反应的性质 将它们与原始的未折叠的形式分开 研究了

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.