Phd Position - Combining Selective Dnp-Nmr And Site-Specific Labeling To Study Active Sites Of...

Universities and Institutes of France

France

September 20, 2022

Description

  • Organisation/Company: Idex université Grenoble Alpes
  • Research Field: Biological sciences › Biology Chemistry › Biochemistry
  • Researcher Profile: First Stage Researcher (R1) Recognised Researcher (R2) Established Researcher (R3) Leading Researcher (R4)
  • Application Deadline: 20/09/2022 00:00 - Europe/Brussels
  • Location: France › Grenoble
  • Type Of Contract: Temporary
  • Job Status: Full-time
  • Offer Starting Date: 01/12/2022
  • IDEX PROJECT TITLE: GlycoAlps

    SUBJECT TITLE: Combining selective DNP-NMR and site-specific labeling to study active sites of large biomolecular assemblies: the case of bacterial endosulfatases.

    RESEARCH FIELD: Chemistry, biological sciences

    SCIENTIFIC DEPARTMENT (LABORATORY'S NAME): Modelization and Exploration of Materials, MEM (UMR 9001 CEA-UGA) + Occasionnally: NMR-Bio World Trade Center, 5 place Robert Schuman 38025 Grenoble

    DOCTORAL SCHOOL'S: Ecole Doctorale ESCV

    SUPERVISOR'S NAME: Sabine HEDIGER et Rime KERFAH

    Description of subject:

    The PhD student will be involved in a highly interdisciplinary research project at the frontiers of Chemistry, Biology and Physics. The aim is to continue the development of selective hyperpolarization NMR approaches for their application to large biomolecular complexes. Dynamic Nuclear Polarization (DNP) has emerged as a promising hyperpolarization technique for solid-state NMR. Nevertheless, for biomolecules, the spectral resolution is often strongly impacted by the use of cryogenic temperature. The MEM DNP team recently introduced a new methodology, called Selective DNP (SelDNP), which allows recovering high-resolution DNP spectra for specific parts of the protein 1. This targeted DNP-NMR approach 2 combines paramagnetic tagging (radical- functionalized ligand, spin label, paramagnetic metal ion, etc.) and differential spectroscopy. It was demonstrated to reveal the carbohydrate- binding site of the protein LecA (12 kDa). Thanks to the expertise of NMR-Bio in advanced specific isotopic spin labeling (e.g. 2H, 15N, 13C, 13CH3, etc.) and of our collaborators from the University of Island in radical chemistry, we propose to extend this emerging and promissing SelDNP methodology to large biomolecules. As a proof of concept, we will first focus on a chondroitin sulfate 4-endosulfatase (ENDO-4sulfatase, 58 kDa) expressed by Prevotella oris, a bacteria of the human gut microbiota. Sulfatases are important and yet poorly understood enzymes, which play a key role in regulating the sulfation states of many important substrates, including complex cell-surface polysaccharides. Beyond the mentioned consortium, the project will also involve collaboration with R. Vivès (IBS) specialized in sulfatases. Ultimately, the development of the SelDNP methodology should provide new solutions for investigating protein/glycan interactions and glycoenzymes. 1 I. Marin-Montesinos, D. Goyard, E. Gillon, O. Renaudet, A. Imberty, S. Hediger and G. De Paëpe, Chem. Sci., 2019, 10, 3366. 2 D. Gauto, O. Dakhlaoui, I. Marin-Montesinos, S. Hediger and G.De Paëpe, Chem. Sci., 2021,12, 6223.

    Missions:

    The PhD student will be involved in a highly interdisciplinary research project at the frontiers of Chemistry, Biology and Physics. The aim is to continue the development of selective hyperpolarization NMR approaches for their application to large biomolecular complexes. Dynamic Nuclear Polarization (DNP) has emerged as a promising hyperpolarization technique for solid-state NMR. Nevertheless, for biomolecules, the spectral resolution is often strongly impacted by the use of cryogenic temperature. The MEM DNP-NMR team recently introduced a new methodology, called Selective DNP (SelDNP), which allows recovering high-resolution DNP spectra for specific parts of the protein 1. This targeted DNP-NMR approach 2 combines paramagnetic tagging (radical-functionalized ligand, spin label, paramagnetic metal ion, etc.) and differential spectroscopy. It was demonstrated to reveal the carbohydrate-binding site of the protein LecA (12 kDa). Thanks to the expertise of NMR-Bio in advanced specific isotopic spin labeling (e.g. 2H, 15N, 13C, 13CH3, etc.) and of our collaborators from the University of Island in radical chemistry (Prof. Snorri Siggurdsson's group), we propose to extend this emerging and promising SelDNP methodology to large biomolecules. As a proof of concept, we will first focus on a chondroitin sulfate 4-endosulfatase (ENDO-4sulfatase, 58 kDa) expressed by Prevotella oris, a bacteria of the human gut microbiota. Sulfatases are important and yet poorly understood enzymes, which play a key role in regulating the sulfation states of many important substrates, including complex cell-surface polysaccharides. Beyond the mentioned consortium, the project will also involve collaboration with R. Vivès (IBS) specialized in sulfatases. Ultimately, the development of the SelDNP methodology should provide new solutions for investigating protein/glycan interactions and glycoenzymes. 1 I. Marin-Montesinos, D. Goyard, E. Gillon, O. Renaudet, A. Imberty, S. Hediger and G. De Paëpe, Chem. Sci., 2019, 10, 3366. 2 D. Gauto, O. Dakhlaoui, I. Marin-Montesinos, S. Hediger and G.De Paëpe, Chem. Sci., 2021,12, 6223.

    Main activities:

    The PhD student will be involved in both protein production/labelling (under the supervision of NMRBio) and their investigation with solid-state NMR and DNP within the MEM laboratory. Notably he/she will be trained to become autonomous on the DNP-NMR spectrometer, including specific DNP sample preparation, handling of low temperature MAS and microwave irradiation, recording and processing of DNP-NMR experiments, and their assignment and interpretation. In addition, complementary access to the French national network of NMR facilities (IR- RMN) will be requested if necessary for the project (https: // www. ir- rmn.fr/en/).

    Structure description

    MEM is one of the Research Units of the Interdisciplinary Research Institute of Grenoble (IRIG), which brings together fundamental research in biology, health, nanosciences, cryotechnologies and new technologies for energy and the environment. The MEM laboratory groups together a unique and coherent set of state-of the art techniques for material exploration, such as electronic microscopy, synchrotron X-ray diffraction, neutron diffraction, advanced simulations and theory, as well as solid-state and hyperpolarized NMR (DNP). Research at MEM covers the development of these state-of-the-art techniques coupled to advanced material characterization and applications. Within MEM, the DNP-NMR group of the Magnetic Resonance laboratory focuses on the development of state of the art DNP-NMR instrumentation and methodology to push the limits of sensitivity and resolution of the technique, in order to address challenging systems ranging from material to life sciences, including glycosciences. Our laboratory is equipped with two 400 MHz/263 GHz DNP spectrometers, operating at ~100 K and down to ~20 K (in-house instrumental development, quasi-unique prototype worldwide). The laboratory is equipped with standard wet-lab capability and has a workshop (oscilloscopes, VNA) to work on probes etc. Since we are located inside the MINATEC nanocharacterization platform we also have privileged access to state-of-the- art microscopes (SEM, TEM), XPS, etc. In addition, our laboratory hosts three research-oriented NMR spectrometers (500 / 400 / 200 MHz) equipped for solid- and solution-state NMR, alongside capabilities for diffusion studies, HRMAS, in-operando and 1.3 mm fast MAS probes for low field paramagnetic NMR. NMR-Bio is a translational contract research organization founded by researchers from IRIG, specialized in the specific isotopic labelling of proteins for their investigation by NMR.NMR-Bio offers products for labelling as well as custom recombinant protein production and NMR services, but is also deeply involved in R&D programs to expand its expertise in protein NMR and develop new labeling protocols. Grenoble is one of the major cities in Europe for research with a large international scientific community. In addition, Grenoble has a large international student population. It is a very pleasant city to live in, and is known as the “Capital of the Alps” with easy access to great skiing and hiking. It is also only 2 hours' drive to the Mediterranean Sea, Italy, or Switzerland. Grenoble, Lyon, and Geneva airports are nearby and permit straightforward international travel.

    Team description (N+1 and colleagues) :

    The solid-state DNP-NMR team (https: // nmr-dnp-grenoble.net/home/) is led by Gaël De Paëpe and composed of 3 researchers (S. Hediger, S. Paul and G. De Paëpe,). The group currently hosts 3 PhD students and 2 Postdocs. Their research aim at improving the sensitivity and resolution of DNP-NMR and includes instrumental and methodological developments as well as application to challenging systems ranging from advanced functional materials (cellulose nanocrystals, ZnO, pillared graphene, perovskite etc.) to biomolecules and cells. Sabine HEDIGER (CNRS Research Director) is an expert in solid-state NMR with particular focus on methodological developments in DNP and their application to biomolecular systems. Gaël DE PAËPE (CEA Research Director, head of the Magnetic Resonance lab) is an expert in solid-state NMR and DNP, including instrumental and methodological developments as well as application to material sciences and biomolecules. Elodie Crublet is the CEO of NMR-Bio, which includes 2 full time project managers: Rime Kerfah and Rida Awad. NMR- Bio researchers have PhDs in Biochemistry and structural biology.

    Other:

    Safety: work in the presence of magnetic fields and cryogenic fluids; standard chemical and biological risks.

    Funding category: Contrat doctoral

    PHD title: Combining selective DNP-NMR and site-specific labeling to study active sites of large

    PHD Country: France

    Offer Requirements Specific Requirements

    Previous formation, diplomas: Master in structural biology, chemistry or physical chemistry (including courses in biochemistry/molecular biology).

    Trade skills/expertise: background in biochemistry and biophysics required. Strong interest in structural biology and physical chemistry (including NMR spectroscopy). Practical knowledge in protein production and overexpression recommended. Basic knowledge in (solid-state) NMR spectroscopy would be an asset.

    Personal skills: strong motivation to work in a highly interdisciplinary and international environment. Good teamwork and communication skills in English, both oral and written.

    Eligibility criteria

    Applicants must hold a Master's degree (or be about to earn one) or have a university degree equivalent to a European Master's (5-year duration).

    Applicants will have to send an application letter in English and attach:

    - Their last diploma

    - Their CV

    - A short presentation of their scientific project (1-2 pages max)

    - At least one letter of recommendation from previous research supervisors.

    Contact Information
  • Organisation/Company: Idex université Grenoble Alpes
  • Organisation Type: Public Research Institution
  • Website: https: // edu.univ-grenoble-alpes.fr/
  • Country: France
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