title: A new NMR technique to probe protein-ligand interaction.
abstract: Non covalent grafting of proteins on affinity phases is a very common approach for isolation, purification and re-concentration of tagged proteins. Many biophysical studies are conducted on these grafted proteins (surface plasmon resonance, quartz crystal microbalance, etc.) showing that the integrity and function of the protein is usually maintained. However, NMR studies of such samples were not undertaken so far, due to the broadening observed on this kind of heterogeneous samples. We present here the use of the HR-MAS technology to obtain 2D NMR spectra of the MAGI-1 PDZ2/6 protein domain, C13-labeled, tagged with a His-tag and grafted on a Nickel affinity resin. We optimized the C13 Methyl SOFAST HMQC experiment allowing important gains in terms of signal-to-noise. The gain comes from the gathering of proton magnetization from the resin material to the protein under study. Several methyl signals from the unstructured C-terminal tail, which is involved in the binding of the PDZ domain to C-terminal peptides of its partners, were observed and measured. The interaction of the bound PDZ domain with cognate peptides was monitored using <500μg of protein sample. A response proportional to the peptide Kd is obtained, indicating that the method can be used to rapidly and efficiently monitor protein-ligand interactions.
authors.affiliation: IGBMC, CNRS UMR 7104, 1 rue Laurent Fries BP10142, 67404 Illkirch, France; NMRTEC, 850 Boulevard S. Brant, Bioparc B, 67400 Illkirch, France.
authors.forename: J M P
authors.initials: JM
authors.lastname: Viéville
authors.affiliation: IREBS, UMR-7242, Oncoprotein Team, ESBS, 300 Boulevard S. Brant, BP 10413, 67412 Illkirch, France.
authors.forename: S
authors.initials: S
authors.lastname: Charbonnier
authors.affiliation: IGBMC, CNRS UMR 7104, 1 rue Laurent Fries BP10142, 67404 Illkirch, France.
authors.forename: P
authors.initials: P
authors.lastname: Eberling
authors.affiliation: NMRTEC, 850 Boulevard S. Brant, Bioparc B, 67400 Illkirch, France.
authors.forename: J-P
authors.initials: JP
authors.lastname: Starck
authors.affiliation: IGBMC, CNRS UMR 7104, 1 rue Laurent Fries BP10142, 67404 Illkirch, France; NMRTEC, 850 Boulevard S. Brant, Bioparc B, 67400 Illkirch, France. Electronic address: delsuc@igbmc.fr.
authors.forename: M-A
authors.initials: MA
authors.lastname: Delsuc
mesh_terms: D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D008024:Ligands; D010455:Peptides; D011506:Proteins
keywords: Grafted protein; HR-MAS NMR; MAGI1 PDZ2/6; Protein–ligand interaction; SOFAST HMQC