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.
