STRUCTURE OF A CAPSID ASSEMBLY BY 1H-DETECTED MAS NMR
Protein structure determination is key to the detailed description of many biological processes. The critical factor that would allow general application of magic-angle spinning (MAS) solid-state NMR to this end is improvement in sensitivity and resolution for as many nuclear spins as possible. This was achieved at the CRMN with the detection of resolved 1H resonances in protonated proteins by increasing MAS rates to frequencies of 100 kHz and above. For large proteins and assemblies, ultrafast spinning narrows spectral resonances better than Brownian motion on which solution NMR relies, removing a fundamental barrier to the NMR study of large systems. This was exploited in a milestone publication to determine the de novo structure of a 28-kDa protein dimer in a 2.5-MDa viral capsid assembly.
STRUCTURE OF A SURFACE SITE BY DNP-SENS NMR
The ability to detect and characterize the three-dimensional (3D) structure of molecules at the atomic scale, through the introduction of a range of physical methods, has transformed molecular, biological and materials science over the past 50 years. However, if the species of interest is located on a surface, structure determination has so far not been possible. In this context, the demonstration that the full 3D structure of a Pt complex anchored on an amorphous silica surface could be determined by DNP SENS has been landmark achievement. The 200-fold increase in the NMR sensitivity of the surface fragment provided by DNP enabled the implementation of a series of multidimensional and multi-nuclear NMR correlation experiments providing quantitative structural restraints. Several 13C-15N and 29Si-15N distances were obtained under rotational echo double resonance (REDOR) experiments. The result, in combination with EXAFS, was a detailed structure for the surface complex, determined with a precision of 0.71 Å.
DEVELOPMENT OF FRAGMENT-BASED INHIBITORS OF PROTEINS
Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. A fragment-based drug discovery approach using NMR, X-ray crystallography and structure-based compound optimization was used to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds circumvents the disadvantages of the existing cyclophilin inhibitors, and has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections. NMR-based strategies for the screening of small molecular-weight compounds (so-called fragments) against purified proteins samples represent today the most attractive application of NMR in pharmaceutical industry.
Hyperpolarization by dissolution dynamic nuclear polarization has made ultra-sensitive magnetic resonance become a reality and has triggered the development of a plethora of promising applications in spectroscopy and imaging. Unfortunately, some severe limitations severely restrain the widespread use of this method, amongst which the experimental complexity, the need for trained personnel, and the exuberant price. We are developing solutions for a broad democratization of hyperpolarization by enabling transport over long distances, so as to bring molecules directly in a hyperpolarized state at the point of use. We have recently pioneered a new concept in which transport was demonstrated in some micro-crystalline formulated 13C-labelled molecules (see for example here the extension of the lifetime of hyperpolarized pyruvate from minutes to tens of hours). We are currently working on a new approach where arbitrary molecules, with arbitrary formulations (ranging from crystals to glass and frozen solutions), can be equally hyperpolarized at state-of-the-art levels exceeding P(13C) > 40%, and further stored for hours in view of transport to a remote point of use.
NEW METHODS FOR CONFORMATIONAL DYNAMICS OF BIOMOLECULES
Describing conformational plasticity of biomolecules is essential to understand their detailed biophysics and connect it to their biological function. However, the complexity of these processes often prevents such descriptions. We actively developed novel experimental and computational methods, both for RNA or proteins to access the description to those key biophysical processes. In particular, we described how a chaperone protein can dynamically perturb the conformational landscape of its substrate to trigger its folding in absence of external source of energy.
CUTTING-EDGE INTERNATIONAL TRAINING
Over the past years, the CRMN has been involved, as coordinator (pNMR) or as partner (ZULF-NMR) in two Initial Training Networks (ITN), which are specific H2020 MSCA actions supporting PhD and post-doctoral positions and allows training-through-research via a system of exchange between young researchers and laboratories. Integral to these research-based training programs has been a series of workshops, practical training courses, international conferences, and outreach actions, located at the different sites. Overall, these actions have been not only providing doctoral training for 4 PhD students at the CRMN and 16 more in different EU countries, but also spreading the essential know-how between various academic EU groups and extending it to those segments of industry that either supply NMR spectroscopy equipment or use NMR, e.g., in the pharmaceutical industry. The international visibility of the CRMN with respect to these training activities is further witnessed by three MSCA incoming fellowships hosted during the last five years. Thanks to these projects and the resulting international visibility, four postdoctoral researchers who worked at the CRMN have recently obtained professorships in prestigious universities (University of California at Santa Barbara, Stockholm University, Max-Planck Institute, Warsaw University).
The instrumentation of the platform has been enriched recently with the acquisition of several new probes (first worldwide 1.3 mm DNP and 0.7 mm MAS probes), of a Bruker Avance NEO console for the 1 GHz spectrometer, of a benchtop Bruker EMX Nano EPR spectrometer, and last but not least, the installation of two polarizers for dissolution DNP, one of which installed in the CRMN spectrometer hall, which offers the possibility to perform hyperpolarized solution NMR at very high magnetic fields.
As part of the IRICE program (Installations de Recherche et d’Innovation Centrées Entreprises) of the Auvergne-Rhône-Alpes Region, the CRMN has been funded a 3.2 M€ project ("An NMR platform centered on excellence in the service of the innovation in chemistry and health "), to undertake a major upgrade of the NMR platform (consoles and last generation probes). Supported by competitiveness clusters, notably Axelera and Lyonbiopôle, by research institutes such as Bioaster and Canceropôle Clara, and a first circle of companies in the Region, the project aims to provide regional companies with integrated access to our NMR platform to meet their growing needs in terms of analytical characterization in the fields of chemistry and health, and more generally will help to increase the NMR's visibility to regional socio-economic actors, its integration into the innovation ecosystem, and the transfer of technology.