Woodburn, Lauren Frances; (2024) Multidomain protein folding on the ribosome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Many proteins in the human proteome consist of multiple domains which must fold co-translationally, lest they risk misfolding and aggregation. Inter-domain contacts trap misfolded conformations which prevent proteins like the tandem repeat protein (TRP), filamin, from functioning as intended; this can cause a multitude of degenerative diseases. Multidomain misfolding is integrally linked with the relative paucity of data to inform this co-translational folding (CTF) process, as misfolding renders multidomain systems experimentally challenging to study in vitro. We sought to establish exactly the mechanisms by which multiple TR domains are synthesized during translation, in a protein which is theoretically an obligate cotranslational folder, and how misfolding is averted in situ. This investigation entailed design of a system to study co-translational TRP folding, examining the vectoral synthesis of a model Ig-like TRP as it emerges from the ribosome using an integrated approach: nuclear magnetic resonance spectroscopy (NMR), cryo-electron microscopy (cryo-EM), biochemistry and molecular dynamics (MD) simulations were each applied to resolve a different facet of CTF. Specifically, we investigated the length-dependent folding of two filamin domains, FLN4 and FLN5, when tethered to the ribosome by varying lengths of a third, FLN6, using 1H-15N, 1H13C-methyl, and 19F NMR spectroscopy, Molecular Dynamics (MD) and protein engineering to probe structure and dynamics (Chapter 2). We uncovered a dynamic flux between two domains which facilitates folding and generates earlier structure formation during CTF, while stabilising co-translational intermediate states. We then employed protein engineering to further probe this interface between domains to discover two intermediates stably coexist in tandem without risk of misfolding, mediated in part by residue specific interactions. The FLN5 domain was therefore capable of folding on the ribosome after an unfolded FLN4, and enabled CTF intermediates to persist in both FLN4 and FLN5 until the end of full-length Filamin (domains 1-6) synthesis (chapter 3). Furthermore, we probed the interactions between the nascent chain and the ribosome, resolving a binding event with the vestibule which acts to delay folding onset: a biochemical assay (PEGylation) was designed with the intent to measure thermodynamic stability of FLN5 on and off the ribosome, which determined the energetic influence of this ribosome-binding event on the stability of NCs. Finally, we employed cryo-EM to identify the binding site at the ribosome exit tunnel and generated maps which highlighted the conformational heterogeneity in the vestibule (chapter 4). This investigation has provided a detailed view of coordinated folding during synthesis on the ribosome, and resolved dynamic events using a multidisciplinary approach which develops on 30 years of work investigating multidomain protein folding.

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