Cotranslational folding of HemK C-terminal domain

by Jan Daberger
Doctoral thesis
Date of Examination:2023-02-13
Date of issue:2023-03-17
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Jörg Enderlein
Referee:Dr. Alexander Stein
Referee:Prof. Dr. Kai Tittmann
Referee:Dr. Sonja Lorenz
Referee:Dr. Alex Faesen
Persistent Address: http://dx.doi.org/10.53846/goediss-9786

 

 

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Abstract

English

The 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