Roeselová, Alžběta; (2024) Cotranslational maturation of a multi-domain oligomeric protein at the ribosome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Protein folding in the cell begins during protein synthesis at the ribosome in the presence of multiple molecular chaperones. How the ribosome and chaperones influence folding remains poorly understood. This is especially true for cotranslational maturation of multi-domain and oligomeric proteins which have been generally underrepresented in protein folding studies. Here, we optimized a strategy to study protein synthesis intermediates - ribosome nascent chain complexes - with hydrogen/deuterium exchange mass spectrometry. This allowed us to obtain a detailed structural description of the cotranslational folding pathway of a multi-domain protein, β-galactosidase. We show that while some β-galactosidase domains fold immediately upon synthesis, folding of other regions is delayed until synthesis and folding of C-terminally encoded domains. Moreover, the ribosome affects β-galactosidase maturation in multiple ways. It directly interacts with the nascent chain (NC) near the exit tunnel, has a far-reaching destabilizing effect, and delays onset of cotranslational assembly. Next, we show that multiple molecular chaperones engage β-galactosidase cotranslationally. Near the ribosome surface, Trigger factor recognizes compact folding intermediates exposing extensive non-native regions and prevents DnaJ and DnaK binding to NCs. Further away from the ribosome, DnaJ and DnaK promiscuously bind non-native chains until translation termination. Neither of the chaperone systems destabilize cotranslational folding intermediates. GroEL can also interact with RNCs via the inside of its main cavity, resulting in NC destabilization. Subsequent partial encapsulation by GroES promotes NC refolding. GroEL binding to NCs is limited to sites not occupied by Trigger factor and is in direct competition with DnaK/J. In summary, by utilizing a complex model protein, this thesis contributes to our understanding of how multi-domain proteins mature on the ribosome and how the chaperone network modulates cotranslational folding intermediates and achieves functional redundancy.

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