Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel.

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1-140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63-94 residues. Longer RNCs (apoHmp1-189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.

Mapping Protein-Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome-Nascent Globin Complex.

Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein-ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.

Critical Beginnings: Selective Tuning of Solubility and Structural Accuracy of Newly Synthesized Proteins by the Hsp70 Chaperone System.

Proteins are particularly prone to aggregation immediately after release from the ribosome, and it is therefore important to elucidate the role of chaperones during these key steps of protein life. The Hsp70 and trigger factor (TF) chaperone systems interact with nascent proteins during biogenesis and immediately post-translationally. It is unclear, however, whether these chaperones can prevent formation of soluble and insoluble aggregates. Here, we address this question by monitoring the solubility and structural accuracy of globin proteins biosynthesized in an Escherichia coli cell-free system containing different concentrations of the bacterial Hsp70 and TF chaperones. We find that Hsp70 concentrations required to grant solubility to newly synthesized proteins are extremely sensitive to client-protein sequence. Importantly, Hsp70 concentrations yielding soluble client proteins are insufficient to prevent formation of soluble aggregates. In fact, for some aggregation-prone protein variants, avoidance of soluble-aggregate formation demands Hsp70 concentrations that exceed cellular levels in E. coli. In all, our data highlight the prominent role of soluble aggregates upon nascent-protein release from the ribosome and show the limitations of the Hsp70 chaperone system in the case of highly aggregation-prone proteins. These results demonstrate the need to devise better strategies to prevent soluble-aggregate formation upon release from the ribosome.

Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome.

Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding-site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client-protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome-wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258 Escherichia coli (E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent-exposed than other nonpolar sites, in protein native states. Our findings show that the majority of E. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70-bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70-chaperone-binding traits have evolved to favor Hsp70-assisted protein folding devoid of aggregation.

Protein folding in vitro and in the cell: From a solitary journey to a team effort.

Correct protein folding is essential for the health and function of living organisms. Yet, it is not well understood how unfolded proteins reach their native state and avoid aggregation, especially within the cellular milieu. Some proteins, especially small, single-domain and apparent two-state folders, successfully attain their native state upon dilution from denaturant. Yet, many more proteins undergo misfolding and aggregation during this process, in a concentration-dependent fashion. Once formed, native and aggregated states are often kinetically trapped relative to each other. Hence, the early stages of protein life are absolutely critical for proper kinetic channeling to the folded state and for long-term solubility and function. This review summarizes current knowledge on protein folding/aggregation mechanisms in buffered solution and within the bacterial cell, highlighting early stages. Remarkably, teamwork between nascent chain, ribosome, trigger factor and Hsp70 molecular chaperones enables all proteins to overcome aggregation propensities and reach a long-lived bioactive state.

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins.

This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57-83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.