Solid-state NMR methods for protein and protein complexes. – Bio-ssNMR

The research proposal was dedicated to the development of new solid-state NMR methodology and strategies for the structural and dynamical characterization of challeging biological assemblies. We followed three main approaches to meet the challenges of this research project. The first one consists in the development of new, sophisticated NMR experiments to remove the main barriers in terms of spectral resolution and sensitivity. This part of the project capitalized on unique NMR equipments, including the first worldwide 1 GHz spectrometer (the most powerful high-resolution NMR spectrometer in the world, installed in Lyon in 2009). The second approach concerned the development of state-of-the-art computational data analysis protocols for consistent and accurate collection of conformational restraints, which represents the second critical step in structure determination of solid proteins. The structural investigation of large protein assemblies from the DNA replisome were then tackled by applying the experimental and computational methods developed above.

We have first designed new multi-dimensional solid-state NMR experiments to improve the resolution and sensitivity of protein spectra under ultra-fast magic angle spinning. In parallel, we have demonstrated that the use of high magnetic fields (800 and 1000 MHz) and ultra-fast magic angle spinning of the samples (60 kHz) allows narrow 1H line widths to be achieved from large fully-protonated proteins. We have also started gathering information about protein-protein interactions in the replisome, a highly complex, multi-domain, molecular machine that carries out the replication of DNA.

On the methodological side, the results obtained in this project will push forward solid-state NMR studies of biologically relevant systems in general, and will contribute to pushing back the current size limits.
The project on the replisome assemblies will produce results of outstanding significance to the broader scientific community: no structure of a replisome (the complex molecular apparatus that carries out the replication of DNA) in any of its functional states is yet known. In the absence of a complete replisome structure, the preliminary characterization of several subassemblies by NMR would potentially aid to construct a model of a complete replisome. This knowledge will have several wide-ranging implications, for example allowing the exploitation of those interactions as targets for antibacterial agents.

The project was a considerable success, leading to 10 publications in top-level chemistry journals, (with 2 more already submitted, and 1 more in preparation), 3 book chapters, 12 invited conferences and 16 promoted talks at international meetings.

Solid-state NMR (ssNMR) is applicable to a wide range of chemical and biological problems that cannot be addressed by solution NMR or X-ray crystallographic methods. Steadily ongoing methodological developments combined with tremendous advances in probe and spectrometer hardware have led to a variety of strategies for resonance assignment, paving the way to the first 3D structure determinations of a wide range of samples at atomic resolution, ranging from inorganic frameworks, catalysts to membrane proteins and fibrils.
Solid-state NMR does not suffer from molecular weight limitations (which are affecting its solution counterpart), and it can be applied to non-crystalline samples. Therefore, this spectroscopy is uniquely positioned to answer key questions about chemistry and structure of macromolecular assemblies, which, because of their size and structural flexibility, are often difficult to characterize. Several important problems remain however to be solved before ssNMR is ready to cope with challenging solid biochemical assemblies, and many methodological developments are still expected in this fast evolving field. The proposed project includes the development of innovative experimental ssNMR methodologies and their application to the structural investigation of biologically relevant proteins and protein complexes.
Sensitivity and spectral complexity first limit solid-state NMR investigations on molecules of high-molecular weight. Capitalizing on recent methodological developments initiated in our group, we will thus focus on the design of innovative solid-state NMR strategies aiming at removing the current barriers in terms of sensitivity and resolution. As an essential step in characterizing the architecture of complex assemblies consists in probing protein-protein interfaces, new solid-state NMR strategies will also be developed to map solvent exposure and to deduce inter-domain contacts.
Although three-dimensional structures of a few crystalline and non-crystalline biomolecules have been recently reported, no standard protocol exists for the structure determination of solid proteins. State-of-the-art computational protocols for solid-state NMR data analysis will thus be developed as well as novel strategies for sequence-specific resonance assignments of proteins in the solid-state. Both the experimental and computational developments will be conducted on model protein systems already available in our laboratory (microcrystalline GB1, Ubiquitin and SOD), to establish a proof of principle, before being applied to more challenging protein complexes as described below.
The ultimate goal of the project is to apply solid-state NMR spectroscopy to investigate the structure of an outstandingly challenging multi-domain protein complex, namely the functional alpha:epsilon:theta sub-assembly of the E. coli DNA polymerase III. This large macromolecular assembly (161 kDa) contains the catalytic core of the enzyme, and cannot be analyzed with atomic resolution by any other technique. We plan to reconstitute the complex from the separately overproduced and purified components, and to precipitate them, in order to characterize their molecular structure by NMR in the solid state. Sample preparation will be carried out in collaboration with Prof. Dixon (Australia), a world-recognized expert in DNA replication. Both uniformly and specifically 13C/15N isotopic labeled protein samples at strategically selected sites will be considered. Various experimental protocols will be investigated to obtain immobilized samples suitable for solid-state NMR investigations. Resonance assignment of the NMR spectra will then be performed using state of art methodology on both the isolated and re-assembled domains. In a last step, the interactions between the various sub-units will be probed by using several NMR methods, including chemical shift mapping, to potentially derive structural model of the protein assemblies.