Deciphering the structural mechanisms of RAF kinase regulation

You will join a dynamic, multidisciplinary team with on-site access to cutting-edge facilities in cryo-EM, X-ray crystallography, NMR, biochemistry, biophysics, molecular biology and cell biology.  The project is part of an international collaboration in which the student will take part.

Mission:

We are looking for a motivated M2 student in protein biochemistry and structural biology to decipher the molecular mechanisms of RAF kinase signalling associated with human cancer.

Project:

The RAF kinase family is a key component of the RAS-ERK signalling pathway controlling cell growth and proliferation. Hyperactivation of this pathway is a hallmark of cancer, with RAF mutated in ~8% of tumors. Although the RAS-ERK pathway is heavily targeted in the clinic, resistance mechanisms limit drug efficacy. Improving our understanding of the RAS-ERK pathway regulation is therefore crucial to develop new treatments.

We previously showed how RAF kinases are regulated by KSR pseudokinases (Lavoie*, Sahmi*, Maisonneuve* et al. Nature 2018) and by the scaffolding proteins CNK and HYP (Maisonneuve*, Sahmi* et al. Nature Structural & Molecular Biology 2024). More recently, we obtained strong preliminary results showing that RAF activation is controlled by additional regulatory proteins whose mechanisms of action remain poorly understood. Our aim is to characterize the structural mechanisms by which these novel regulatory proteins modulate RAF activation.

The candidate will join Dr. Maisonneuve’s team (CBMN, UMR 5248, IECB) and will be involved in protein expression in bacteria and insect cells, protein purification and functional characterisation using biochemical and biophysical approaches such as NMR, BLI, SPR and fluorescent polariation. The student will also contribute to the structural characterization of higher-order RAF complexes  by cryo-electron microscopy (cryo-EM).

Methodology:

• Molecular biology (site-directed mutagenesis of expression plasmids).
• Protein expression in bacteria and insect cells (established in the lab).
• Protein purification by affinity, ion exchange and gel filtration chromatography (established in the lab).
• Structural characterization of protein complexes by single particle cryo-EM.
• In vitro functional/biophysical assays of protein-protein interactions (NMR, BLI, SPR or fluorescent polarization, etc).

• Master 2 or 5^th year engineering student
• Previous experience in protein expression and purification is strongly preferred.
• Good background in biophysics (BLI, SPR, FP) and structural biology is encouraged