Chaperones are essential to the co-translational folding of most proteins. However, the principles of co-translational chaperone interaction throughout the proteome are poorly understood, as current methods are restricted to few substrates and cannot capture nascent protein folding or chaperone binding sites, precluding a comprehensive understanding of productive and erroneous protein biosynthesis. Here, by integrating genome-wide selective ribosome profiling, single-molecule tools, and computational predictions using AlphaFold we show that the binding of the main E. coli chaperones involved in co-translational folding, Trigger Factor (TF) and DnaK correlates with "unsatisfied residues" exposed on nascent partial folds - residues that have begun to form tertiary structure but cannot yet form all native contacts due to ongoing translation. This general principle allows us to predict their co-translational binding across the proteome based on sequence only, which we verify experimentally. The results show that TF and DnaK stably bind partially folded rather than unfolded conformers. They also indicate a synergistic action of TF guiding intra-domain folding and DnaK preventing premature inter-domain contacts, and reveal robustness in the larger chaperone network (TF, DnaK, GroEL). Given the complexity of translation, folding, and chaperone functions, our predictions based on general chaperone binding rules indicate an unexpected underlying simplicity.

Conformational control of nascent chains is poorly understood. Chaperones are known to stabilize, unfold, and disaggregate polypeptides away from the ribosome. In comparison, much less is known about the elementary conformational control mechanisms at the ribosome. Yet, proteins encounter major folding and aggregation challenges during translation. Here, using selective ribosome profiling and optical tweezers with correlated single-molecule fluorescence, with dihydrofolate reductase (DHFR) as a model system, we show that the Escherichia coli chaperone trigger factor (TF) accelerates nascent chain folding. TF scans nascent chains by transient binding events, and then locks into a stable binding mode as the chain collapses and folds. This interplay is reciprocal: TF binding collapses nascent chains and stabilizes partial folds, while nascent chain compaction prolongs TF binding. Ongoing translation controls these cooperative effects, with TF-accelerated folding depending on the emergence of a peptide segment that is central to the core DHFR beta-sheet. The folding acceleration we report here impacts processes that depend on folding occurring cotranslationally, including cotranslational protein assembly, protein aggregation, and translational pausing, and may be relevant to other domains of life.

Protein complexes are pivotal to most cellular processes. Emerging evidence indicating dimer assembly by pairs of ribosomes suggests yet unknown folding mechanisms involving two nascent chains. Here, we show that co-translational ribosome pairing allows their nascent chains to ‘chaperone each other’, thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity during early translation stages is found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly-competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.

The role of ribosome-bound chaperones and nascent chain interactions in protein biogenesis holds many open questions. This thesis provides new insights into the intricate mechanisms governing co-translational folding and complex assembly. By combining in vivo genome-wide screening results from selective ribosome and disome selective profiling, conducted by our collaborators B. Bukau and G. Kramer at Heidelberg University, with our in vitro single-molecule force spectroscopy and correlated confocal imaging results, we not only gained a better understanding of the general prevalence, but also of the mechanistic details of these cellular processes.

We investigated how Trigger Factor (TF), the only ribosome-associated chaperone in bacteria, influences nascent chain folding of a single-domain protein. We show for the first time that TF accelerates folding on the ribosome. This process is regulated by translation, with the emergence of key peptide segments dictating TF’s ability to compact nascent chains and stabilize partial folds. Beyond chaperone-modulated folding, we also explored how ribosome cooperation drives protein complex formation. Using the intermediate filament lamin as a model, we demonstrate that ribosome proximity enables nascent chains to ‘chaperone each other,’ facilitating coiled-coil formation while preventing misfolding. Notably, when early interactions between nascent chains are inhibited or delayed, they become trapped in misfolded states and are no longer assembly-competent. We further examined the role of timing in nascent chain interactions, using the BTB domain as a model to study the challenging formation of intertwined dimers during translation. We identify a translation-driven temporal control mechanism that ensures proper dimerization. This process opens otherwise inaccessible folding-assembly pathways, bypassing unproductive monomeric states.

Taken together, we have managed, through the powerful combination of in vivo and in vitro approaches, to gain new perspectives on how cells ensure faithful protein biogenesis.

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70.