Cotranslational folding of HemK C-terminal domain
von Jan Daberger
Datum der mündl. Prüfung:2023-02-13
Betreuer:Prof. Dr. Marina Rodnina
Gutachter:Prof. Dr. Marina Rodnina
Gutachter:Prof. Dr. Jörg Enderlein
Gutachter:Dr. Alexander Stein
Gutachter:Prof. Dr. Kai Tittmann
Gutachter:Dr. Sonja Lorenz
Gutachter:Dr. Alex Faesen
Diese Datei ist bis 12.02.2024 gesperrt.
EnglischThe correct folding of proteins is essential for the survival of the cell. Despite the importance of the question, it remains largely unclear how proteins, in particular large multidomain proteins with mixed α-helical and β-strand structures, fold cotranslationally. In this study, we chose the C-terminal domain of E. coli enzyme HemK as the model protein to study folding in a fully reconstituted in vitro translation system. Here, we present the folding pathway of the protein step-by-step employing force profile assay using the SecM arrest peptide in combination with time-resolved and steady-state photo-induced electron transfer and Förster resonance energy transfer measurements. This combination allows us to analyze the folding pattern both inside and outside of the polypeptide exit tunnel of the ribosome and to assess the stepwise compaction of the nascent chain. The N-terminal β-hairpin of the C-terminal domain starts to fold inside the polypeptide exit tunnel and undergoes structural rearrangements before emerging from the ribosome. As the nascent peptide emerges from the ribosome, it folds sequentially, starting from the first β-hairpin, which could serve as the folding nucleus for the following C-terminal part adopting a Rossmann fold. The folded domain remains highly dynamic on the ribosome and the final compaction into the native structure occurs after its release from the ribosome.
Keywords: Cotranslational folding; Ribosome; In vitro translation