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PhD in Membrane Protein Biochemistry (M/F)

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Français - Anglais

Date Limite Candidature : mercredi 6 octobre 2021

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General information

Reference : UMR7099-MANZOO-001
Workplace : PARIS 05
Date of publication : Wednesday, September 15, 2021
Scientific Responsible name : Zoonens
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 November 2021
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly

Description of the thesis topic

Membrane proteins (MPs) are challenging research targets due to many difficulties affecting all steps of sample preparation. Notably, extraction from the native lipid environment with detergents is a critical step because low-affinity protein-lipid interactions can be lost, turning out possibly in misleading data. A challenge for the next decade is to study MPs together with their surrounding native lipids [1]. One of the thematics led in the laboratory of physical-chemical biology of membrane proteins (IBPC, Paris) is the development of amphipathic polymers, called amphipols, to diversify the toolbox available for solubilizing and stabilizing MPs along with their native lipids [2].
In the frame of an ANR project, in collaboration with the channel receptor laboratory (Institut Pasteur, Paris) and the laboratory of bioorganic mass spectrometry (IPHC, Strasbourg), we will test the benefit of amphipols in preserving the protein-lipid interactions by using as model MPs several members of the pentameric ligand-gated ion channel (pLGIC) superfamily, which play a key physiological role in neuronal communication, and are known to be exquisitely sensitive to lipid interactions. Previous works on the nicotinic acetylcholine receptor (nAChR) extracted from Torpedo marmorata and reconstituted in liposomes [3] and on the bacterial homolog ELIC [4] have shown that cholesterol and anionic lipids are required for the channels to activate, but the exact nature and the contribution of endogenous lipids to pLGICs activity remain to be characterized.
During this PhD thesis, we plan to test an amphipol library to solubilize a series of membrane preparations or cell lines expressing pLGICs (E. coli, HEK 293 cells and insect S2 cells) and use the most promising polymers for protein purification. Co-purified lipids will be extracted and analyzed by mass spectrometry. The native state, i.e. the correct stoichiometry of receptors, and the sample homogeneity will be characterized by different approaches (size exclusion chromatography, negative-stain electron microscopy, and mass photometry). To evaluate the impact of extraction conditions on protein stability, we will also assess the thermal stability of selected samples using thermal-shift assay. Finally, to directly monitor the active state, we will use a technique using ethidium bromide, which specifically binds within the open channel of the Torpedo nAChR, yielding a large increase in fluorescence intensity [5]. Comparison of data in intact membrane, in detergent or in amphipols will allow us to assess whether allosteric transitions are preserved, and whether particular allosteric states are thermodynamically favored.
References:
1. Ratkeviciute G, Cooper BF, Knowles TJ: Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation. Biochemical Society Transactions 2021, 49:1763–1777.
2. Marconnet A, Michon B, Le Bon C, Giusti F, Tribet C, Zoonens M: Solubilization and Stabilization of Membrane Proteins by Cycloalkane-Modified Amphiphilic Polymers. Biomacromolecules 2020, 21:3459–3467.
3. daCosta CJB, Medaglia SA, Lavigne N, Wang S, Carswell CL, Baenziger JE: Anionic lipids allosterically modulate multiple nicotinic acetylcholine receptor conformational equilibria. J Biol Chem 2009, 284:33841–33849.
4. Tong A, Petroff JT, Hsu F-F, Schmidpeter PA, Nimigean CM, Sharp L, Brannigan G, Cheng WW: Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel. Elife 2019, 8.
5. Sun J, Comeau JF, Baenziger JE: Probing the structure of the uncoupled nicotinic acetylcholine receptor. Biochimica et Biophysica Acta (BBA) - Biomembranes 2017, 1859:146–154.

Work Context

The Laboratory of Physical Chemical Biology of Membrane Proteins is located in the 5th disctrict of Paris (http://umr7099.ibpc.fr/). It gathers biologists, physicists and chemists who are interested in the structure, structural dynamics and physical chemistry of membrane proteins, either in membrane mimetic systems (liposomes or nanodiscs), or in classical detergent solutions or alternative surfactants such as amphipols. Currently, there are 19 permanent staff, 3 fixed-term contracts and 4 PhD students.

Constraints and risks

The project will be performed in close collaboration with the Channel Receptor Laboratory located at Pasteur Institute in Paris (https://research.pasteur.fr/fr/team/channel-receptors/). Experiments in the two laboratories will be performed.
In terms of risk, a radioligand binding assay is considered, but all safety conditions will be applied.

Additional Information

To apply, please provide a CV, a cover letter and ideally a letter of recommendation.
Any application with a level of study higher than a PhD will not be accepted.

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