THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYMATIC MECHANISMS

这个实验室从事蛋白质结构和蛋白质的研究。 一种蛋白质分子的机制，它是以随机的 螺旋，可以折叠成特定的二级和三级结构，而不需要 任何外部帮助。研究的主要对象是猪胃蛋白酶原，一种 相对分子质量为39,630的单体蛋白质，在pH值范围内稳定 在6到8.5之间。在pH 6以下，胃蛋白酶原通过蛋白分解激活自身 失去了最初的44个氨基酸，产生了一种具有酶活性的 蛋白质，胃酶。胃酶只有在pH低于6时才稳定。这两种蛋白质都是 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，胃蛋白酶原可以重新折叠到 它的正常结构，当回到自然条件下，而胃酶 不能。我对这种折叠反应的机理很感兴趣 顺序的改变对两者行为的影响 蛋白质。使用诸如紫外线、圆二色和 荧光光谱学，加上化学修饰和 多肽化学，天然物种和未折叠物种的结构 被刻画出来了。使用快速运动技术，例如停流 和T-跳跃，中间和部分折叠的形式已在 折叠反应，它们的结构已经部分确定，并且 化学反应的性质，将它们与天然和 调查展开的表格。

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. 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 unfolded forms investigated.