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Reference : UPR2301-NELMOR-001
Workplace : GIF SUR YVETTE
Date of publication : Tuesday, June 30, 2020
Scientific Responsible name : Nelly Morellet
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 October 2020
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
Cyclodipeptide synthases (CDPS) produce cyclodipeptides and complex derivatives, diketopiperazines (DKP), which constitute a broad class of natural products synthesized by microorganisms and possessing remarkable pharmacological properties such as antibacterial, antiviral or anticancer. To do this, CDPS divert aminoacyl tRNAs (aa- tRNAs) from their canonical role in protein synthesis by ribosomes.
The thesis project consists in carrying out a structural study of the interaction of CDPS (cyclodipeptide synthases) and their substrates, the aa- tRNAs, and thus identify the CDPS residues responsible for substrate specificity. This project is part of a more global project, funded by the ANR, which will use the acquired structural data to perform engineering on CDPS and obtain variants with modified specificities that can, for example, use substrates containing non-canonical amino acids. The aim of this global project is to produce, through an ecological biosynthesis process, different DKPs with high therapeutic potential. The ANR project is led by Muriel Gondry (Peptide Biosynthesis and Gene Expression (LBPEG)), Institut des Sciences du Vivant Frédéric-Joliot, I2BC, Gif sur Yvette), in partnership with Jean-Baptiste Charbonnier for the X-ray crystallography part (Institute of Integrative Cell Biology, I2BC, Gif sur Yvette), Nelly Morellet and Ewen Lescop (Institut de Chimie des substances Naturelles, Gif sur Yvette) for the NMR part and Eric Jacquet in charge of the high throughput quantitative PCR platform (Institut de Chimie des substances Naturelles, Gif sur Yvette).
- The thesis will take place in the three laboratories presented above. The aim will be to study the interaction between CDPS and aminoacyl mini-RNAs.
- The PhD student will carry out the production of proteins in Muriel Gondry's team (I2BC, Gif sur Yvette), enriched in 15N and 15N-13C and 15N-13C-2D, by overexpression in Escherichia Coli and purification by a system of three successive chromatographic columns.
- The best solution conditions will be achieved in the high throughput quantitative PCR platform under the supervision of Eric Jacquet (ICSN, Gif sur Yvette), using thermal shift tests that quantify the change in thermal denaturation temperature of a protein under different conditions of pH, salt concentrations, type of buffers and additives (glycerol ...).
- Crystallization tests will be carried out under the responsibility of Jean-Baptiste Charbonnier in order to obtain structures by X-ray crystallography of CDPS:aa-mini-RNA complexes. Measurements of biophysical interactions will be carried out to evaluate the affinities of different aa-mini-RNAs for the CDPS studied by switchSense, calorimetry and thermophoresis in particular.
- NMR studies will be carried out under the responsibility of Nelly Morellet (ICSN, Gif sur Yvette). Initially the attribution of HN, CO, Ca and Cb resonances of CDPS proteins enriched in 15N and 13C (248 amino acids) will be performed using the classical 3-dimensional NMR experiments, HNCO, HNCA, HNCA, HNCOCA, HNCACB and HNCOCACB. Depending on the quality of the spectra, the recording of these experiments will be performed on proteins also enriched in deuterium on the side chains.
- The 1H-15N HSQC spectra will then be used to map on the protein the interaction zones of aminoacyl mini-RNAs by observing chemical shift variations in the HSQC spectra of the proteins.
- Thesis writing and papers
The Institut de Chimie des Substances Naturelles is a Clean Research Unit of the CNRS (French National Center for Scientific Research) whose research focuses are chemistry and biology. It is part of the University of Paris-Saclay which, since 2015, has brought together research and teaching establishments in the south of Paris. The ICSN is located on the Gif-sur-Yvette campus and has about 125 staff members, 76 of whom are permanent employees spread over 3 buildings.
Within the Department of Chemistry and Structural and Analytical Biology, the staff member recruited will join the biology and structural chemistry team consisting of 5 IT, 8 CH and 2 CDD/doctoral students. He/she will be placed under the hierarchical authority of the team leader. The officer will work directly with Nelly Morellet and in collaboration with Ewen Lescop.
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