Interdomain functional dynamics of phosphoglycerate kinase studied by single-molecule FRET

The thesis reflects the steps followed to perform reliable single-molecule FRET (smFRET) experiments on phosphoglycerate kinase (PGK) from yeast. The main text is essential and divided in short chapters in order to relieve the reading. All technical details, demostrations, long descriptions, protocols, figures, and tables are reported in the appendix for the reader more interested in a deeper understanding. The first part encompasses Chap.3 to Chap.9 and is dedicated to the establishment of reliable single-molecule fluorescence measurements. Here, it is condensed the Ph. D. experience in a manual-like form for newcomers of the field willing to perform single-molecule experiments from scratch. Since high quality and controlled labeled samples are a fundamental prerequisite for single-molecule fluorescence measurements, this topic is discussed in Chap.3. The theoretical background for the comprehension of singlemolecule detection in solution is given in a compact and consistent form in Chap.4. The basics about the three principal experimental approaches needed to perform single-molecule fluorescence measurements are presented in Chap.5, Chap.6 and Chap.7. The data treatment of the smFRET experiments is explained in Chap.8 and is mainly based on the works of Gopich and Szabo, and Seidel and co-workers. The procedure applied to determine the set of parameteres required for the analysis of the data is found in Chap.9. Here, a new strategy for the measurement of precise relative quantum yields on the confocal microscope is reported. Finally, in the last chapter (Chap.10) the whole knowledge is applied to study the interdomain dynamics of PGK. Here, the protein motions are modeled with an elastic network simulated under a random Multiparticle Collision Dynamics (rMPC) approach