A new NMR technique to probe protein-ligand interaction

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.