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(se abrirá en una nueva ventana)) 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.
