Report
Teaser, summary, work performed and final results
Periodic Reporting for period 1 - RIBOFOLD (Ribosome Processivity and Co-translational Protein Folding)
Teaser
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...
Summary
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. During its lifetime in the cell a protein can undergo structural rearrangements due to binding to its ligand and partner proteins, but its initial fate depends on the correct structure attained during the pioneering folding event on the ribosome. 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. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. The assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding. Finally, 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.
Work performed
The main focus of the reporting period was to establish new methods to screen for co-translational folding intermediates. In addition to time resolved Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET), we established two new approaches. One new method now established in our lab is the arrest peptide-mediated force profile assay (FPA). FPA is a biochemical method to monitor force generation events at various stages of cotranslational folding. As the peptide folds within the exit tunnel, it exerts mechanical tension, which is transmitted over the length of the nascent chain to the peptidyl transferase center of the ribosome. In the absence of any additional mechanical force, a short translational arrest peptide, SecM, is sufficient to stall translation. A high-tension mechanical pulling force originating from nascent protein folding, can alleviate the SecM stalling and restart translation. FPA can identify folding events at a codon resolution, although the nature of these folding intermediates has to be determined using alternative methods.
Another new method now established in out lab to study protein folding is fluorescence correlation spectroscopy (FCS). FCS can provide unique insights into protein dynamics down to nanosecond time resolution. To fully harness the FCS potential and study the local conformational fluctuations of peptide chains, FCS can be further combined with fluorescence quenching by photoinduced electron transfer (PET-FCS). This approach was successfully applied to study protein folding and dynamics in solution. PET requires short-range interactions (van der Waals contacts, <1 nm), which allows to specifically monitor the local structural fluctuations of the protein chains. Using PET-FCS to monitor cotranslational folding intermediates on the ribosome could provide insight into their structural dynamics and detect rapid local fluctuations between the different conformations of the nascent chain.
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 – 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. In both cases, the pace of translation is rate-limiting for protein folding. The structure of the CspA folding interemediates is currently under study by cryo-electron microscopy (in collaboration with X. Agirrezabala and M. Valle, Bilbao, Spain). The experiments on the effect of ribosome-associated protein biogenesis factors PDF and MAP on the rate of co-translational folding are ongoing as planned.
The results obtained so far are described in two papers and presented on nine 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, which attracts about 5000 participants in total. We are developing 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.
Final results
The main technical achievement beyond the state of the art is establishing the FCS technique and developing the dedicated data analysis that reveals the dynamics of protein nascent chains attached to the ribosome. 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. The groundbreaking result of these studies is that the pathway of co-translational protein folding can differ dramatically from that is solution and that on the ribosome proteins fold by vectorial assembly of consecutive structural elements, rather than by concerted folding observed in solution.