18 months Post-doctoral position at Univ. Grenoble Alpes / CEA, France, on DNP-enhanced solid-state NMR

18 months Post-doctoral position at Univ. Grenoble Alpes / CEA, France, on DNP-enhanced solid-state NMR

Methodological developments towards in-cell DNP

We are looking for a motivated postdoc to work with Sabine Hediger and Gaël De Paëpe on developing hyperpolarized solid-state NMR (DNP, Dynamic Nuclear Polarization) for the study of proteins in cells. More information about our group at CEA Grenoble / Univ. Grenoble Alpes and a list of recent publications can be found here: https://nmr-dnp-grenoble.net. The project will be done in collaboration with the group of Aurélien Deniaud, at CEA Grenoble (https://www.cbm-lab.fr/en/Pages/MetOr/Presentation.aspx), expert in cell biology, copper homeostasis in mammals, and biochemistry of copper binding proteins.

Context of the project:

Cellular applications of solid-state NMR are associated with major challenges: low sensitivity and resolution, need for isotopic labelling strategies able to suppress the large cellular background. Dynamic Nuclear Polarization (DNP)has emerged as a viable route to significantly improve the sensitivity of solid-state NMR, including for biomolecular and in-cell applications. In addition, DNP experiments are performed at low temperature (about 100 K or lower), which offers the additional advantage to protect the cell integrity. Some groups have started to investigate the potential of the technique towards in-cell applications, but most approaches so far suffer from a lack of selectivity to detect low protein amounts under the large background signal of the cell, or required a highly deuterated cellular environment. In addition, spectral resolution is impaired by conformational heterogeneity induced by sample freezing.

Project:

We propose to solve the problems of resolution, sensitivity and selectivity associated with in-cell DNP experiments by developing an innovative approach based on the combination of bio-orthogonal chemistry and selective DNP (SelDNP). The methodology will be applied on mammalian cells for the study of Atox1, a copper (Cu) chaperone involved in Cu homeostasis.

Based on preliminary results, the methodology will be developed in stages, first on purified proteins, and then on the protein in mammalian cells. The first step will be to optimize experimental conditions for selective DNP (SelDNP) on Atox1 and to determine the best targeting strategy (including polarizing agent) that allows detecting sub-micromolar concentrations in cell lysates. This knowledge will then be used to target and detect Atox1 directly in the mammalian cell, using bio-orthogonal chemistry to introduce the spin label for SelDNP. SelDNP is a methodology developed over the last few years in our group. It enables the production of high-resolution DNP-enhanced NMR subspectra specific to a predefined region of the protein, determined by the position of the spin label. Demonstrated so far only on purified proteins, the methodology is very promising for targeting a biomolecule in the crowded environment of the cell, but as well for removing the cell background signal, which become significant at low protein concentrations.

Requirements and application:

Applicants are expected to have a doctoral degree in liquid-state and/or solid-state biomolecular NMR spectroscopy. Knowledge about MAS-DNP will be considered as a plus. The candidate will mainly work in NMR and DNP, but depending on his/her expertise in biochemistry, he/she may assist in protein production and labeling.

The successful candidate will be recruited for 18 months. Deadline for application is end of May. Interested candidates are welcomed to send an email to: sabine.hediger@cea.fr

Local environment:

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, 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.

Sabine Hediger Interdisciplinary Research Institute of Grenoble CEA Grenoble 17 rue des Martyrs 38054 Grenoble Cedex 9 France Tel.: +33 4 38 78 65 79 Fax : +33 4 38 78 50 90 Email : sabine.hediger@cea.fr ________________________________________