MIF NMR Chemical Shift Perturbation Mapping

MIF, as a cytokine, is implicated in several disease classes, including cancer, autoimmunity, and viral infection, making it an attractive target for therapeutic development. Many biophysical techniques can provide estimates of binding affinity, which are helpful as compound screening techniques and invaluable in validating biological assay results. Among these techniques, Heteronuclear Single Quantum Coherence spectroscopy (HSQC) NMR offers significant advantages that justify its inherent technical challenges, including its high sensitivity, robustness to false positives, and the ability to determine binding affinities at micromolar to millimolar concentrations. Furthermore, by mapping the amino acids whose NMR chemical shift resonances are perturbed by compound binding onto the 3D structure of MIF, researchers can infer the binding site of the compound. Here, we present an isotopic ¹⁵N labeling technique for MIF. We also present conditions for obtaining high-quality ¹⁵N-HSQC NMR spectra and explain how to use chemical shift mapping to determine binding sites and binding affinities.