COMPLEX-fastMAS-NMR · Structure of a large non-crystalline multiprotein assembly by solid-state nuclear magnetic resonance with ultra-fast magic-angle spinning
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2016-01-01 → 2018-01-18
- EU contribution
- €185,076
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Structure of a large non-crystalline multiprotein assembly by solid-state nuclear magnetic resonance with ultra-fast magic-angle spinning
The increasing resistance of pathogens to antibiotic treatment poses a potentially catastrophic threat to public health. There is an emerging need for new drug targets that will be unlikely to bypass by single mutations in bacteria. DNA replication machinery represents a potential candidate: while individual proteins are not conserved among bacteria, replication mechanisms and cascades of interactions are. However, to develop effective drugs, it is crucial to understand not only the atomic structure of these proteins but their weak and transient interactions. In this project, we aimed at the characterisation of large proteins from E. coli replisome using solid-state Nuclear Magnetic Resonance (ssNMR). This rapidly developing method allows to investigate dynamic non-crystalline protein or RNA/DNA samples thus complementing the static information available from the X-ray crystallography or cryo-electron microscopy. The objective of the project was to push the limits of the technique to large biomolecules that can only be obtained in submilligram quantities and/or only with a simple isotope labelling (i.e. without deuteration). To achieve this goal we employed the combination of two unique tools available in the host laboratory: ultra-high magnetic fields and a new generation of probes capable of sample rotation at the so-called magic-angle (MAS) up to 111,111 times per second.
Data: CORDIS, © European Union
Project objective
Extrapolating from protein structure to function is an operation difficult to accomplish, as most functions in the cell are not carried out by single proteins, but by macromolecular complexes containing multiple subunits endowed with specific functions. Despite the many advances in structural and biochemical studies of isolated molecules, a comprehensive portrait of cell biochemistry must thus include insights of the supramolecular network of interactions among the individual constituents. The structure determination of large and dynamical protein ensembles presents a great deal of challenges for X-ray diffraction techniques, and for solution nuclear magnetic resonance (NMR) due to the large size of these objects. Consequently, there is little information available today on the overall organization of the assembly subunits, their interactions, and sometimes their precise function within the cell. High-resolution solid-state NMR (ssNMR) has recently developed as a powerful structural tool for studying structure and dynamics of solid biological samples at atomic resolution. A number of issues remain however to be addressed before ssNMR is ready to cope with large-sized multi-domain functional assemblies.The proposed project aims to capitalize on new concepts recently introduced by the host institution, to implement innovative solid-state NMR methodologies. Sophisticated experimental approaches will be introduced at high magnetic field, in combination with ultra-fast magic angle spinning (MAS), enabling the structure characterization of non-crystalline assemblies that cannot be currently carried out by any other experimental technique, and notably, the determination at atomic level of the structural details that govern protein-protein interactions in functional assemblies. As a benchmark, we will tackle the interaction mode of the ring-shaped hexameric DnaB helicase with its partner DnaC, an assembly that controls the origin of the DNA replication in E. coli.
Original text from CORDIS.
Participants
- CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS · ParisCoordinatorFrance
Links
- View on CORDIS
- DOI: 10.3030/661799
- http://perso.ens-lyon.fr/guido.pintacuda
- https://arquivo.pt/wayback/20201229134524/http://perso.ens-lyon.fr/guido.pintacuda/guidosgroup/Guidos_group.html
Data: CORDIS, © European Union