Weise, A; (2015) Structural investigations of intrinsically disordered polypeptides: biosynthesis on the ribosome and protein misfolding disease. Doctoral thesis , UCL (University College London).

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Abstract

Intrinsically disordered proteins constitute a significant proportion of our proteome and are emerging targets of modern structural and molecular biology through their involvement in a range of functions crucial for the survival of the cell. Their malfunction and aggregation is known in many cases to lead to devastating pathologies including Parkinson’s disease (α-synuclein, αSyn) and amyloid lateral sclerosis (TAR DNA- binding protein, TDP-43). NMR spectroscopy, is a high resolution structural technique that is especially well suited to provide residue-specific information on flexible and unstructured systems, and which is used to investigate the structural and dynamic aspects of αSyn and TDP-43. TDP-43, is a eukaryotic, 440-residue, multi-domain protein with a predicted disordered C-terminal tail (CTD274-414) that initiates the aggregation of TDP-43 and harbours the disease-associated mutations. This thesis describes the development of an expression and purification strategy and the NMR investigations of the structure and dynamics of the CTD274-414 region both in isolation and as it exists within living cells. Using these methods we have initially obtained the complete protein backbone assignment of the CTD274-414 under 8 M urea conditions, which indicated a mostly disordered conformation, serving now as a valuable template to determine the secondary structure propensity of this domain within E. coli lysate. In addition, the aggregation behaviour of the CTD274-414 was investigated using biophysical techniques, which revealed that while the CTD274-414 aggregates appear to have fibrillar morphology, the overall aggregation properties suggest that CTD274-414 does not form typical β-amyloids. The understanding provided by structural biology over recent years of the PD-associated protein αSyn has enabled detailed insights into the conformational dynamics of several forms of this ’chameleon’ protein. Here, a detailed study of the biosynthesis of αSyn using NMR spectroscopic investigations of ribosome-bound nascent chain complexes (RNCs) is presented. By considering the length-dependent emergence, the effects of charge and also the interaction of αSyn RNCs with the TF chaperone, detailed insights of the interactions arising from the interplay between TF, the ribosome and nascent polypeptide chains were gained. These results were used to create a structural model beginning to reveal the specific manner in which disordered proteins interact with the biosynthesis machinery.

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