FOLDING AND DYNAMICS OF A BI-DOMAIN ENZYME

本项目的目的是表征折叠机制的两个- 结构域酶，3-磷酸甘油酸激酶（PGK）。 实验方法 包括基因工程、稳态和时间的结合- 分辨荧光和圆二色性技术。 时间分辨 将进行荧光能量转移测量，以便 确定关键的域内和域间距离对之间的 用外源荧光标记的基因工程半胱氨酸 probes. 基因工程改造的基因组将被用作 在展开过程中监测局部和全局变化的探针， 重折叠过渡 上述方法预计将提供一个 天然、中间和未折叠状态的详细表征， 以及形成的时间顺序（i）个别中学 结构元素，（ii）单个域和（iii）域-域 接口. 这项研究的长期目标是解开信息 包含在指导这个折叠的氨基酸序列中， 酵素

The aim of this project is to characterize the folding mechanism of a two- domain enzyme, 3-phosphoglycerate kinase (PGK). The experimental approach involves a combination of genetic engineering, steady-state and time- resolved fluorescence, and circular dichroism techniques. Time-resolved fluorescence energy transfer measurements will be carried out in order to determine the key intra- and inter-domain distances between pairs of genetically-engineered cysteines, labeled with extrinsic fluorescent probes. Genetically-engineered tryptophans will be used as intrinsic probes to monitor local and global changes during the unfolding and refolding transitions. The above approach is expected to provide a detailed characterization of the native, intermediate and unfolded states, as well as the time-sequence of the formation of (i) individual secondary structure elements, (ii) individual domains and (iii) the domain-domain interface. The long range goal of this study is to unravel information contained in the amino acid sequence that direct the folding of this enzyme.