| Summary: | It is now well established that proteins can begin to acquire structure in a co-translational manner on the ribosome, in a fundamental process known as co-translational folding (CTF). During synthesis, the emerging nascent chain (NC) faces a choice between fold- ing correctly and misfolding. Proteins that fail to fold correctly can deposit as toxic cellular inclusions, which are found in a range of diseases including Alzheimer's, and Parkinson's diseases, type II diabetes and the serpinopathies. Structural insights into this process have been revealed by applying NMR spectroscopy and cryo-EM to ribosome- nascent chain complexes (RNC) (Cabrita et al., 2016), however little is understood of any mechanisms that underpin co-translational misfolding during biosynthesis. We have thus begun to characterise the biosynthesis of α1-antitrypsin (AAT), which belongs to a serine protease inhibitor superfamily. Disease-associated pathological mutations (e.g. Z-AAT(E342K)) are known to destabilise the protein, rendering it susceptible to misfolding and self-assembly into aggregation-prone polymers. To study how AAT acquires its native fold, we have developed a quantitative cysteine-modification assay (PEGylation) to probe the structural properties of newly synthesised AAT NCs during their emergence and release from ribosomes in a eukaryotic cell-free system. Coupled with kinetic modelling, this approach has enabled us to systematically map the folding pathway of newly synthesised AAT NCs. We show that the NC not only folds to its native state post-translationally, but that it is also prone to significant misfolding shortly after synthesis. By applying quantitative PEGylation and kinetic modelling, we investigated the CTF behaviour of both WT and Z AAT RNCs, by measuring the site-specific solvent accessibilities of ten unique cysteine probe positions. Kinetic modelling reveals that ribosome-bound AAT NC exhibits a conformational exchange between two distinct structural states; this has implications for our understanding of the impact that CTF phenomena have on how NCs are able to fold successfully during biosynthesis.
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