Structural investigation of the folding of an immunoglobulin domain during biosynthesis on the ribosome

Successful protein folding is central to all biological cellular processes with a large portion of the proteome able to begin to acquire its three-dimensional structure in a co-translational manner during its biosynthesis on the ribosome. The vectorial emergence of the nascent polypeptide from the exit tunnel and its attachment to its parent ribosome results in differences between the details of the folding process of isolated polypeptides and that on the ribosome (Introduction). We have developed a detailed strategy to enable the study of co-translational folding using solution-state NMR spectroscopy, the only technique able to characterise this dynamic process at atomic resolution (Chapter 2). Using isotopically-labelled ribosome-nascent chain complexes (RNCs), we have determined a high-resolution, structural description of protein folding on the ribosome via snapshots that mimic the emergence of an immunoglobulin-like domain within a multi-domain protein. Our NMR results reveal the structure and dynamic features of how con- formational space is sampled by a fledgling nascent polypeptide as it converts into its folded state and demonstrate that the entire immunoglobulin domain has to be emerged from the tunnel before native folding is acquired (Chapter 3). These findings contrast with analogous studies of C-terminal truncations of this domain, indicating some exciting differences between folding on the ribosome and that in bulk solution. The ribosome itself is shown to significantly influence the process of folding (Chapter 4); specifically, we use a combination of protein engi- neering and NMR dynamics to reveal residue-specific sites of nascent-chain-ribosome interac- tions of different magnitude of strength. The studies presented are providing the first high-resolution insights of these fundamental processes and they begin to shape our understanding of the energy landscape characterising a nascent chain on the cusp of initiation of folding on the ribosome