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Ribosome Processivity and Co-translational Protein Folding

Periodic Reporting for period 2 - RIBOFOLD (Ribosome Processivity and Co-translational Protein Folding)

Reporting period: 2020-02-01 to 2021-07-31

In every living cell, proteins must adopt specific three-dimensional structures to fulfill their diverse functions. Proteins start to fold while they are being assembled by the ribosome during translation. A newly synthesized protein nascent chain enters the exit tunnel of the ribosome, which provides a narrow space that confines protein folding. Each time the ribosome incorporates an amino acid, the growing peptide moves down the exit tunnel until it emerges from the ribosome. When the synthesis is completed, ready proteins are released from the ribosome and can start to execute their cellular functions. Those proteins that happen to be misfolded are re-folded with the help of the chaperones or degraded by the cellular quality control machinery. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. The pace of translation can change protein folding to produce more or less of the functional peptide form.

Defects in protein folding disturbs the cellular proteostasis, which can result in debilitating diseases. Single amino-acid substitutions can disrupt a protein’s structure in the cell to cause, for instance, cystic fibrosis, sickle cell anemia, cataract, Huntington’s disease, or retinitis pigmentosa. The molecular pathology of these diseases is a perturbation of the native three-dimensional structure leading to a misfolded protein that can no longer execute its function and is prone to aggregation and rapid degradation. Furthermore, mutations in natively disordered proteins, such as α-synuclein, tau protein or amyloid β-peptide, can cause aggregopathies, such as Parkinson’s and Alzheimer’s.

Despite its importance for understanding human diseases, the mechanisms of co-translational folding and the link between the speed of translation and the quality of protein folding are poorly understood. The aim of the RIBOFOLD project is to understand when, where and how proteins emerging from the ribosome start to fold, how the ribosome and auxiliary proteins bound at the polypeptide exit affect nascent peptide folding, what causes ribosome pausing during translation, and how pausing affects nascent peptide folding. To address these questions, we utilize a toolbox of ensemble and single molecule biophysical techniques to monitor translation and protein folding simultaneously at high temporal resolution. We expect that these results will open new horizons in understanding co-translational folding and help understand the molecular basis of many diseases.
We utilized the combination of FRET, PET, PET-FCS, and FPA to study co-translational folding of two selected proteins, the N-terminal alpha-helical domain of HemK, and a single-domain beta-structure protein CspA. We show that compaction HemK starts inside the ribosome as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain's hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.– in contrast to concerted folding of these proteins in solution – their folding on the ribosome is sequential and involves consecutive portions of the proteins as they emerge in the exit tunnel of the ribosome. The nascent chains remain dynamic in the vicinity of the ribosome and rearrange into the stable, compact fold when released from the ribosome. For a β-barrel protein CspA folding also begins by forming a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin that remains dynamic until it is released from the ribosome. The contacts with the ribosome and the structure of the peptidyl transferase center depend on the nascent chain configuration. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome and suggest how protein folding can modulate ribosome activity in elongation and termination. The experiments on the effect of ribosome-associated protein biogenesis factors PDF and MAP on the rate of cotranslational folding are ongoing as planned.

The results obtained so far are described in three papers and two manuscripts that are in revision or submitted for publication and presented on 11 international scientific conferences. While the usual number of participant to such conferences varies between 50 and 300, we were able to present two oral talks at the Biophysical Society Meeting 2020 and European Biophysical Society in 2021, which attracts about 5000 and 1000 participants, respectively. The results were also disseminated through a Youtube webinar (https://www.youtube.com/watch?v=EHWcOoNwzQQ) which collected >600 views so far. We developed a web site and a Twitter showroom to present our research to broader audiences, including not only scientists of different disciplines, but also lay public interested in science.
The main technical achievements beyond the state-of-the-art are two-fold. First, we established the FCS technique and developed the dedicated data analysis that reveals the dynamics of protein nascent chains attached to the ribosome (Liutkute et al., eLife 2020). FCS and PET-FCS have been so far used to study the dynamics of small model proteins. We show that the method is applicable to large macromolecular complexes, which opens the perspective to use this approaches to different macromolecular machines.Second we improved cryo-EM reconstructions to resolve different conformations of nascent peptide inside the exit tunnel of the ribosome (in collaboration with Xabier Agirrezabala and Mikel Valle, Spain). These approaches are used in on-going experiments to solve structures of dynamic conformations of cotranslational folding intermediates.
Switching from α-helical to β-strand conformation upon cotranslational protein folding