Ribosome Processivity and Co-translational Protein Folding

Project Information

RIBOFOLD

Grant agreement ID: 787926

DOI

10.3030/787926

Project closed

EC signature date 2 May 2018

Start date 1 August 2018

End date 31 July 2024

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 482 600,00

EU contribution

€ 2 482 600,00

Coordinated by

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 4 - RIBOFOLD (Ribosome Processivity and Co-translational Protein Folding)

Reporting period: 2023-02-01 to 2024-07-31

Summary of the context and overall objectives of the project

In every cell, proteins must adopt specific three-dimensional structures to function. Protein folding begins co-translationally as the nascent chain moves through the ribosome’s narrow exit tunnel, which constrains folding and, along with ribosomal electrostatic properties, shapes early folding events. Each amino acid addition pushes the growing peptide through the exit tunnel; once complete, proteins are released to perform cellular roles. Misfolded proteins are refolded by chaperones or degraded by quality control systems. Translation speed also influences folding, impacting the yield of correctly folded proteins.

Defects in protein folding disrupt cellular proteostasis and are linked to diseases. Single amino-acid substitutions can destabilize structures, leading to conditions like cystic fibrosis, sickle cell anemia, and Huntington's disease. Such diseases often result from misfolding, aggregation, and loss of function due to structural perturbations. Additionally, mutations in disordered proteins, like α-synuclein and amyloid β-peptide, can lead to aggregopathies such as Parkinson's and Alzheimer's.

Despite the importance of co-translational folding in understanding diseases, the mechanisms behind it remain poorly understood. The RIBOFOLD project addresses these gaps by investigating when and where proteins fold during translation, how ribosome-induced intermediates form, and how translation pauses affect folding. The project also examines how codon usage and translation kinetics impact folding, with the following objectives:

To determine the timing, location, and nature of protein folding events.
To study simultaneous translation effects on interacting proteins.
To explore how ribosome exit port factors influence co-translational folding.
To establish the relationship between translational pausing and folding.
RIBOFOLD aims to enhance understanding of protein quality control and diseases caused by misfolding. Insights from this research could lead to therapeutic strategies and benefit synthetic biology, where correct folding is essential for functional biomolecules.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our team has made significant advances in understanding cotranslational protein folding, focusing on how ribosomes, translation kinetics, and molecular interactions shape folding pathways. Integrating molecular biology, biophysics, and computational modeling, we developed innovative methodologies, gained key insights into folding dynamics, and identified applications relevant to therapeutic development. To capture cotranslational folding in real-time, we created advanced fluorescence-based techniques such as Förster Resonance Energy Transfer (FRET), Photoinduced Electron Transfer (PET), PET Fluorescence Correlation Spectroscopy (PET-FCS), and Force-Profile Assays (FPA). These tools allow high-resolution, single-molecule analysis of nascent polypeptide dynamics on the ribosome, yielding precise data on folding events. Further refinements in labeling nascent chains with bulky fluorescent reporters enhanced both efficiency and fidelity in real-time studies. Our studies on model proteins such as HemK and CspA revealed how the ribosome modulates folding. For example, cotranslational folding of HemK’s N-terminal domain begins within the ribosomal exit tunnel, with alpha-helical segments stabilizing in the confined space and beta-sheet formation commencing in the tunnel’s broader vestibule. These findings underscore the ribosome’s unique energy landscape, which promotes secondary structure formation and prevents misfolding. We also examined translation pauses, influenced by codon usage, tRNA availability, and mRNA structure, which are critical for coordinating the folding of complex proteins. Using synonymous mutations in HemK mRNA, we demonstrated how regulated pauses ensure precise folding, while disruptions lead to misfolding. A predictive algorithm was developed to analyze mRNA sequences for pause sites affecting cotranslational folding, providing a useful tool for studying diseases linked to protein misfolding. Our research on peptide deformylase (PDF), an essential bacterial ribosome-associated protein, showed that PDF scans for formylated substrates on the ribosome and retains deformylated chains until additional processing factors are recruited, potentially shielding nascent chains from premature interactions. This discovery offers a foundation for selective antibiotics targeting bacterial PDF, which is absent in humans, presenting promising implications for combating antibiotic resistance.

RIBOFOLD’s methodologies and findings have broad implications, particularly in translational medicine and antibiotic research. Our techniques, including the “translational ruler,” offer valuable tools for investigating protein misfolding diseases, such as Alzheimer’s, by elucidating how translation kinetics affect proper folding. Additionally, our work on PDF opens new avenues for selective antibiotic development, addressing critical public health needs related to bacterial resistance. The RIBOFOLD project has established our team as a leader in cotranslational folding, providing foundational insights into ribosome-mediated folding and translation kinetics. Our interdisciplinary approach has created potential therapeutic applications in neurodegenerative disease and antibiotic development, with broader impacts extending into computational biology and molecular genetics.

We have disseminated our results through open-access publications, conference presentations, and workshops. Review articles on cotranslational biogenesis factors consolidate our findings and outline frameworks for future research. Additionally, our results were presented in a YouTube webinar (https://www.youtube.com/watch?v=EHWcOoNwzQQ) which has garnered over 1,100,000 views. We also launched a website and a Twitter account to engage both scientific and public audiences, ensuring that RIBOFOLD’s advancements continue to benefit the scientific community and society.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The RIBOFOLD project has advanced beyond the current state of the art by elucidating the ribosome’s active role in cotranslational protein folding, revealing how translation kinetics, ribosomal exit tunnel interactions, and pause sequences shape folding pathways. These methodologies developed in the project facilitate detailed mapping of folding intermediates and dynamics directly on the ribosome, surpassing traditional in-solution studies. Furthermore, our characterization of peptide deformylase (PDF) as a selective target for antibiotics represents a potential breakthrough for combating antibiotic resistance, as PDF is critical for bacterial viability but absent in humans. We anticipate that our results will establish new frameworks for both protein folding research and translational medicine, with tools and knowledge that can benefit molecular biology, disease research, and pharmaceutical development.

Switching from α-helical to β-strand conformation upon cotranslational protein folding