Structural Investigation of an Immunoglobulin Domain on the Ribosome using NMR Spectroscopy

Abstract

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.We have developed a strategy to enable the study of co-translational folding using solution-state NMR spectroscopy, the only technique able to characterize this dynamic process at atomic resolution. 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 multidomain protein. Our NMR results reveal the structure and dynamic features of how conformational 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 possible. These findings contrast with analogous studies of C-terminal truncations of this domain, indicating significant differences between folding on the ribosome and that in bulk solution. The ribosome itself is shown to influence the process of folding; we use a combination of protein engineering and NMR dynamics to describe residue-specific sites of nascent-chain-ribosome interactions of different magnitude of strength, and chemical shift-restrained MD simulations of RNCs to underpin our spectral observations. The studies presented are providing the first high-resolution insights of these fundamental processes and begin to shape our understanding of the energy landscape sampled by a nascent chain on the cusp of initiation of folding on the ribosome.