Periodic Reporting for period 1 - COTCA (Co-Translational Chaperone Action at the Single-Molecule Level)
Période du rapport: 2017-08-01 au 2019-07-31
The mechanism of protein translation by ribosomes has been the focus of recent single molecule investigations. Understanding translation at a single-molecule level is of particular interest to the life sciences and relevant for various degenerative diseases, such as Alzheimer's and Parkinson's. Although protein translation and folding are well studied subjects, cotranslational folding has been proven difficult to observe. How proteins adopt their native structure with efficient fidelity while being synthesized by the ribosome remains largely unexplored. Using optical tweezers we recently measured the mechanics of synthesis and simultaneous folding in real-time, in the absence of chaperones. We found that cotranslational folding occurs at predictable sequence locations, exerting forces on the nascent polypeptide chain. We showed that transient pauses of translation occur in particular locations along the protein sequence, facilitating native secondary structure formation. Several crucial mechanistic questions concerning the effects of chaperones on co-translational folding remain unanswered: How do chaperones such as trigger factor (TF) and the major bacterial heat shock protein 70 (Hsp70/DnaK) affect cotranslational protein folding? What effect do the chaperones have on initial hydrophobic collapse? When and how often do they (un)bind? How do these chaperones assure reliable and fast native folding during protein synthesis? Here, we implemented a combined optical tweezers and laser scanning confocal microscopy study to investigate the effects of chaperones on cotranslational folding in real-time, using the host's instruments, chaperones and collaboration network, as well as my previously developed cotranslational assay and collaboration network. The group of Prof. S. J. Tans at AMOLF with its expertise and experience in single-molecule chaperone investigations was ideally suited for me to pursue this study of cotranslational chaperone activity.
Combining the powerful technique of genome wide selective ribosome profiling (global approach), mastered by our collaborators, with our newly developed high-resolution dual-colour confocal fluorescence single-molecule force spectroscopy assay (fine-grained local approach) has proven to be the perfect marriage. This action has enabled us for the first time to study the intricate transient interactions occurring at the ribosome during the synthesis of proteins in the cell both from a genome-wide bulk-level approach as well as from the single-molecule level. It allowed us to not only resolve a chaperone network at work, it also allowed us to see how protein complexes are forming with the aid of chaperones as the individual subunits of these complexes are being synthesized. Thanks to this fellowship, these highly dynamic processes, previously hidden from view due to the chaotic uncorrelated and fleeting interactions between the myriad of molecules involved, could finally be studied in detail. Furthermore, we have developed a new single-molecule method demonstrating that proteins can fold already deep within the confines of the ribosomal exit tunnel, in a surprising way. Understanding these processes at a molecular level is crucial for understanding the fundamentals of life, how within the crowded environment of the cell often-times large protein complexes can be formed with the folding aid of chaperones without resulting in aggregation of the individual proteins, which has been implicated in a myriad of devastating diseases, such as Alzheimer’s, Parkinson’s disease or laminopathies, such as the Hutchinson-Gilford progeria syndrome. The results of the work conducted in this project has been presented at 11 national and international conferences, reaching a large number of members of the scientific community with wide-ranging backgrounds, from the fields of biophysics to medicine. Several open day events at the host organization and at the European Researchers' Night event in Brussels have served to educate and showcase our research to the general public. The work performed in this project has sparked a new line of research that is still ongoing at the host organization's lab of Sander Tans, including new collaborations and positions in his group that will continue investigating cotranslational phenomena both at a genome-wide and at the single molecule level to disentangle the complex dynamic interactions occurring during protein synthesis in the cell, placing this EU-wide collaborative effort at the top of its field.
With this fellowship we set out to investigate the function of the chaperones trigger factor (TF), the major bacterial heat shock protein 70 (Hsp70/DnaK) during the synthesis of proteins. This work led us to also investigate at the single-molecule level the formation of protein complexes during translation of one of its subunits and how the chaperone TF affects the cotranslational assembly. Along the way we also established new experimental methods to study cotranslational folding at the single-molecule level, resulting in surprising new findings regarding cotranslational folding within the ribosomal exit tunnel. Before the work in this action was conducted it was assumed that only the chaperone TF played a significant role during the synthesis of proteins. Furthermore, trigger factor’s main role was assumed to be as an aggregation shield and an unfoldase, in other word to prevent unwanted interactions with the nascent chain and to keep it from folding too early during synthesis. Our work has shown that TF has other mode of actions. DnaK is thought to act mainly post-translationally, but our work has shown that it is involved in cotranslational folding for a surprisingly high number of substrates. For this chaperone network TF appears to be firmly in the driver’s seat, stabilizing compact non-native folding intermediates, that are further stabilized by DnaK upon TF’s release from the ribosome-nascent chain complex. Cotranslational assembly has not been investigated before using single-molecule techniques. Being the first to describe this process at the single-molecule level will also have a great impact in a number of different fields. Dissemination of our findings is ongoing and we expect a number of high-impact publications to result from this work. The expected fallout from this project as a whole will be of great benefit to Europe's knowledge-based economy and society. Due to the relevance of protein folding, translation and protein complex assembly in several diseases, especially neurological disorders, the project has the potential to also benefit the wider public directly by driving research and innovation in the medical field.