Time-Resolved Single Molecule FRET Studies on Folding/Unfolding Transitions and on Functional Conformational Changes of Phosphoglycerate Kinase

In the presented work unfolding/refolding transitions and functional conformational domain movements of the multi-domain protein, pholphoglycerate kinase (PGK), were investigated. First a study on the unfolding and refolding kinetics using CD spectrocopy was conducted. The kinetics of guanidine hydrochloride (chevron-plot) and heat (eyring-plot) induced unfolding were studied. In this way it was shown, that PGK unfolding is a two state transition. The very high $\textit{m}_{U}$-value indicated, that PGK id highly sensitive to chemical denaturants and that upon unfolding the eccessible surface area is signiticantly increased. From the thermodynamic parameters it can be concluded that in the transition state a lot of native state interactions are broken and hence the transition state represents a less ordered confguration. Refolding is initiated by a collapse into an intermediate state, which already contains secondary structure elements. The subsequent slow refolding step takes place via another intermediate. In a second step a suitable FRET system was established. Therefore three cysteine double mutants were prepared that allow to measure the unfolding/refolding of PGK N-domain, of C-domain and the inter-domain distance changes upon unfolding. The corresponding single systeine mutants were prepared for required control measurements. the respective double cycteine mutants were prepared for required control measurements. The respective double cysteine mutants were labeled with a FRET pair Alexa 488/647. Single molecule FRET measurements were performed on an inverted confocal microscope in a highly diluted sample. By measuring one molecule at the time it was possible to identify and to alanyze distibutions of different sub-populations. With this type of sample the unfolding transition of the inter-domain interface and the N-domain was studied by empolying FRET efficiency histograms. In both cases early unfolding intermediates, and thus putative late folding intermediates were found. Also the native state as the starting point for unfolding studies was subjected to a deteiled investigation. It wa found, that some salts induce a compaction of the native state of PGK and it was shown that the capability of a salt to compact the native PGK follows the Hofmeister series. From the hydrodynamic radius such changes are hardly visible. The functionally relevant native state dynamics was another point of investigation. PGK undergoes a hinge-bending movement to bring the two substrates MgATP and 3-PG in a close vicinity with respect to ech other. By single molecule FRET in particular fast interdomain dynamics were observed that would ensure efficient catalysis