Vujkovic Bukvin, Ivana; (2023) Structures and targeting of the nascent polypeptide folding pathway on the ribosome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

As nascent proteins emerge from their parent ribosomes, the majority of these begin to fold co-translationally. The details of folding on the ribosome differ from those of isolated protein folding in dilute aqueous solution through the vectorial synthesis (4-20 residues/sec), the presence of the large, 2.4 MDa ribosome particle through which the nascent chain emerges and likely several other factors (Chapter 1). Indeed, the emerging nascent protein samples conformational space dynamically in its quest for its three-dimensional structure and undergoes both transient interactions with itself and the external surface of its ribosome. These interactions are poorly understood in molecular detail despite their importance in modulating the folding process. In order to provide accurate structures of these dynamic complexes, we have developed and utilised the use of paramagnetic relaxation enhancement NMR (PRE-NMR) on ribosome-bound nascent chain complexes (RNCs) representing translation snapshots during biosynthesis (Chapter 2). These measurements are enabling the collection of extensive intrachain distance information of an immunoglobulin-like domain on the ribosome. Furthermore, site-specific labelling of the ribosome surface has allowed additional intermolecular distance measurements between the nascent chain and the ribosomal surface. The distance restraints obtained via PRE-NMR have been used to provide atomistic models at the onset of nascent chain folding, revealing initial contacts formed within the co-translational folding nucleus and providing detailed insights into the extent to which the ribosome modulates nascent protein folding via sequence-specific interactions (Chapter 3). We exploited our structural understanding of co-translational protein folding to develop a strategy to specifically target states explored only during biosynthesis. We show that the nascent chain can be selectively targeted as part of the RNC complex at equilibrium, and we are progressing towards developing an antibody binder targeting the Huntingtin nascent polypeptide during its biosynthesis on the ribosome (Chapter 4).

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