FOLDING AND DYNAMICS OF A BI-DOMAIN ENZYME

这个项目的目的是描述两个-的折叠机制- 结构域酶，3-磷酸甘油酸激酶(PGK)。实验方法 涉及基因工程、稳态和时间的结合- 分辨荧光和圆二色谱技术。时间分辨率 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 探测器。基因工程色氨酸将被用作内在的 用于在展开期间监视局部和全局变化的探测器 重新折叠过渡。上述方法预计将提供一种 原生态、中间态和展开态的详细表征， 以及(I)个体次级结构形成的时间顺序 结构要素，(二)个别领域和(三)领域--领域 界面。这项研究的长期目标是解开信息 包含在指导这一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.