Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy

The folding of many proteins can begin during biosynthesis on the ribosome and can be modulated by the ribosome itself. Such perturbations are generally believed to be mediated through interactions between the nascent chain and the ribosome surface, but despite recent progress in characterising interactions of unfolded states with the ribosome, and their impact on the initiation of co-translational folding, a complete quantitative analysis of interactions across both folded and unfolded states of a nascent chain has yet to be realised. Here we apply solution-state NMR spectroscopy to measure transverse proton relaxation rates for methyl groups in folded ribosome–nascent chain complexes of the FLN5 filamin domain. We observe substantial increases in relaxation rates for the nascent chain relative to the isolated domain, which can be related to changes in effective rotational correlation times using measurements of relaxation and cross-correlated relaxation in the isolated domain. Using this approach, we can identify interactions between the nascent chain and the ribosome surface, driven predominantly by electrostatics, and by measuring the change in these interactions as the subsequent FLN6 domain emerges, we may deduce their impact on the free energy landscapes associated with the co-translational folding process

Nascent chains can form co-translational folding intermediates that promote post-translational folding outcomes in a disease-causing protein

During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z’s misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol

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

How Does the Ribosome Fold the Proteome?

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures