Structure of a large non-crystalline multiprotein assembly by solid-state nuclear magnetic resonance with ultra-fast magic-angle spinning

Informazioni relative al progetto

COMPLEX-fastMAS-NMR

ID dell’accordo di sovvenzione: 661799

DOI

10.3030/661799

Progetto chiuso

Data della firma CE 31 Marzo 2015

Data di avvio 1 Gennaio 2016

Data di completamento 18 Gennaio 2018

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 185 076,00

Contributo UE

€ 185 076,00

Coordinato da

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - COMPLEX-fastMAS-NMR (Structure of a large non-crystalline multiprotein assembly by solid-state nuclear magnetic resonance with ultra-fast magic-angle spinning)

Periodo di rendicontazione: 2016-01-01 al 2017-12-31

Sintesi del contesto e degli obiettivi generali del progetto

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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

We evaluated the strenghts of the method (in terms of resolution and sensitivity) on a number of proteins, ranging from small model ones (GB1, MNEI) to complex and dynamic protein assemblies: fibrils (HET-s), viral capsids (AP205, HIV-1) or oligomers from E. coli replisome (DnaB), under >100 kHz magic-angle spinning and at the highest magnetic field commercially available. Despite a certain loss in resolution of 1H resonances, we lean towards the full protonation of proteins as a simpler and more general method of sample preparation for solid-state NMR compared to approaches based on extensive deuteration. We demonstrated that large protonated proteins such as those from E. coli replisome can be immobilised by sedimentation, and studied with ssNMR without a significant penalty in resolution. We developed new sensitive methods for the efficient resonance assignment in proteins and ribonucleic acids, which are applicable thanks to improved spectral properties at ultrafast MAS conditions. We also proposed methods to report on proton-proton proximities that allow a determination of three-dimensional structures of fully protonated proteins. New labelling schemes were devised to assist structure modelling of assemblies. These methodological advances were communicated to broad audience of general chemistry and biology journals, and are already exploited in NMR groups world-wide.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

We demonstrated the feasibility of structural and interaction studies with ssNMR with > 100 kHz MAS and ultrahigh magnetic fields on fully protonated (13C,15N-labelled) proteins and RNA, with sample amount requirements decreased to less than 0.5 mg. The high sensitivity of the novel 1H-detected techniques allowed site-specific NMR resonance assignment not only in the protein backbone but also in the side-chains, and within only a few weeks of experimental time. The information available from side-chains, previously inaccessible, enables one to determine structural models of proteins with an extensive aid of automated tools. This progress in the ssNMR opens new avenues for atomic-level investigations of proteins and nucleic acids which are critical to cellular processes. Overall, it may contribute to efforts to tackle the drug-resistant pathogens that globally affect public health.

Pushing limits of Solid-state NMR for a deeper insight into large biomolecules