There was no better date than today, February 11, the International Day of Women and Girls in Science, to congratulate Lucile Guyot on her Ph.D on membrane proteins, in particular GPCRs (A2AR) and ion channels, with the evaluation of amphiphilic polymers as an alternative to more conventional detergents. A thesis via a CIFRE grant in partnership with the IMPReSs (Integral Membrane Proteins Research and Services) platform and NovAliX under the direction of Dr Renaud Wagner (UMR7242, Biotechnologie et Signalisation Cellulaire Plateforme IMPPReSs) and Dr Gilbert Bey, NovAliX.
The jury was unanimous in recognizing the quality of the presentation and the manuscript as well as the discussion.
The thesis focused on the evaluation of amphiphilic polymers for the extraction, purification and characterization of membrane proteins of therapeutic interest. This work consisted in developing and/or adapting protocols for the production and purification of two model membrane proteins using detergents. These 2 proteins are the adenosine A2A receptor (a G protein coupled receptor) and the TRPV4 ion channel. If the protocols previously developed in the IMPReSs laboratory for the AA2A protein were already well established, this was not the case for TRPV4 for which the production but especially the purification protocol was entirely developed. The purified proteins were then analyzed by cryo-electron microscopy and after many optimizations, a 3D reference structure with a resolution of about 6 Å could be obtained.
The second part of this work consisted in evaluating the efficiency of different amphiphilic polymers to extract the two model proteins from yeast membranes. After identification of the efficient polymers and development of the extraction conditions, an important part of this work consisted in the optimization of the purification protocol.
The last part of this project focused on the characterization of the purified particles, in comparison with the proteins purified in detergent. For AA2A, we performed protein-ligand interaction studies using two techniques, competitive radioactive ligand binding assays and surface plasmon resonance (SPR), which allowed us to measure the affinity of polymer-purified proteins for various agonist and antagonist ligands. For TRPV4, we have performed structural studies but optimizations are still needed to be in conditions compatible with cryo-EM studies.
We wish Lucile a bright and fruitful future as a scientist and wish her to be an inspirational example for future generations of women and girls in science.