JCJC SVSE 8 - JCJC - SVSE 8 - Biochimie, biologie moléculaire et structurale

Cellular Solid State NMR Spectroscopy of membrane proteins from Corynebacteriales – CoryneNMR

Submission summary

Corynebacteriales is a suprageneric actinomycetes group that includes corynebacteria, mycobacteria, nocardia, rhodococci. These Gram+ bacteria are important in several ways: pathogenic mycobacterial species, including Mycobacterium tuberculosis and M. leprae, are known to cause severe infectious diseases in humans. In addition, some corynebacteria are of considerable economic benefit like Corynebacterium glutamicum, the world’s largest producer of glutamate and lysine. This group of microorganisms presents a highly specific cell envelope whose molecular and supramolecular architectures, notably the mycolic acid-containing outer membrane (or mycomembrane), are still poorly understood, and which play crucial roles for survival and interactions with host cells and transport of drugs and nutriments.

Due to very recent advances in solid state NMR, it is now possible to study the structure of membrane proteins associated to the mycomembrane, and of their surroundings, directly in situ on intact cells or envelope preparations. This relies on optimised expression and labelling strategies and on advanced solid state NMR experiments. Dynamic Nuclear Polarisation (DNP) can be used as well in order to enhance sensitivity. This is a major breakthrough, since it eliminates the need for tedious procedures of heterologous expression and reconstitution, and most importantly, since it allows studying native, functional architectures.

The present proposal aims at developing this new exciting NMR opportunity in the field of Corynebacteriales research.

The various scientific tasks we have defined are:
- Optimization of membrane protein expression and isotope labelling: establish a general expression protocol enabling the production of an arbitrary protein target from Corynebacteriales; exploring different expression vectors, C. glutamicum strains and growth and labelling conditions.
- Preparation of ssNMR sample and their characterisation: obtain highly reproducible, well-defined and well characterised biochemically cellular-like preparations.
- Application of existing ssNMR methodologies for their dedicated use on cellular preparations: ssNMR strategies and pulse sequences, DNP based experiments, efficient computing strategies for rapid data interpretation of 2D-3D NMR spectra.
- Investigation of the structure and dynamics of PorA/PorH in cellular-like environments: as a case study, we will solve the structure of the PorA/PorH complex, a porin from the outer membrane which has been largely investigated already in the IPBS.
- Transposition of our protocol to mycobacterial species: explore the possibility to use M. smegmatis as an alternative host expression system.

This project relies of a consortium presenting all the required expertise: besides the coordinator who actively participated in these recent NMR developments in the group Dr M. Baldus in Utrecht in 2008 -2012 (M. Renault et al., PNAS USA, 2012; Angew chemie Int Ed. 2012), it involves two NMR groups (A. Milon, Toulouse and M. Baldus, Utrecht), one group expert in Corynebacteriales biochemistry and microbiology (M. Daffé, Toulouse) and one group expert in biotechnology and molecular biology of Corynebacteriales (L. Eggeling, Jülich).
The project opens an entirely new avenue for functional structural biology in the host laboratory (Institute of Pharmacology and Structural Biology - Toulouse) and will benefit from a total support in terms of equipment and human resources. The project will create a new link between the "Structural Biology and Biophysics" and the "Molecular Mechanisms of Mycobacterial Infections" departments.
Being at the centre of an international collaboration, the project coordinator will have the opportunity to reinforce his scientific independence and international visibility and, if funded and supported by the ANR, she will be in optimum position to apply to an ERC starting grant within 2-3 years.

Project coordination

marie RENAULT (Institut de Pharmacologie et de Biologie Structurale) – marie.renault@ipbs.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IPBS Institut de Pharmacologie et de Biologie Structurale

Help of the ANR 249,999 euros
Beginning and duration of the scientific project: December 2013 - 48 Months

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