TY  - THES
AU  - Bustorff, Nuno
TI  - Folding and structural studies of saccharomyces cerevisiae Phosphoglycerate Kinase
VL  - 282
PB  - RWTH Aachen University
VL  - Dissertation
CY  - Jülich
M1  - FZJ-2024-02574
SN  - 978-3-95806-754-7
T2  - Schriften des Forschungszentrums Jülich Reihe Schlüsseltechnologien / Key Technologies
SP  - xxvi, 126
PY  - 2024
N1  - Dissertation, RWTH Aachen University, 2024
AB  - Proteins are synthesized within cells by ribosomes, and their functionality depend on the correct three-dimensional structure obtained through the process of folding. While the classical understanding of protein folding primarily focused on postsynthesisfolding, recent research has shifted its emphasis toward unraveling the intricacies of folding during synthesis. Of particular interest in this pursuit are multi-domain proteins, which constitute over 70% of proteins in cells. Using classical single-molecule fluorescence resonance transfer efficiency (sm-FRET) studies, the unfolding/refolding transitions of a two-domain yeast phosphoglycerate kinase (yPGK) was explored as a model for multiple domain proteins. To enhance our understanding of a transition within a single-domain of full-length yPGK, I assessed two FRET pair variants within the N-terminal domain. Together with previous data we compared in total six different variants, for which we observed three distinct transitions in both domains: the first segment (positions 1–88 and 202–256, respectively) showed no transition (i.e. no change in the distance), while the second segment (positions 34–135 and 256–290, respectively) underwent a conventional two-state transition. Intriguingly, labeling the complete Rossmann motif (positions 1–135 and 202–290, respectively) revealed a compact intermediate state during GuHCl-induced unfolding in transitory conditions. The detailed understanding of N-terminal domain transitions was essential for comparative analyses with shorter-length proteins synthesized for co-translational folding studies. In order to investigate the ribosomal folding process of yPGK, I employed cryo-electron microscopy (cryo-EM). Four distinct ribosome nascent chain complex (RNC) structures were solved, each representing a nascent polypeptide of a varying length. Most structures depicted nascent chain density outside the ribosomal tunnel, and we observed for the first time the structure of a full-length protein nascent chain attached to the ribosome. In summary, the presented research advances our knowledge of yPGK folding transitions and offers novel routes for studying co-translational folding processes within RNC complexes using sm-FRET and cryo-EM.
LB  - PUB:(DE-HGF)3 ; PUB:(DE-HGF)11
DO  - DOI:10.34734/FZJ-2024-02574
UR  - https://juser.fz-juelich.de/record/1024924
ER  -