Facile detection of protein-protein interactions by one-dimensional NMR spectroscopy

Two methods for detecting protein-protein interactions in solution using one-dimensional (I D) NMR spectroscopy are described. Both methods rely on measurement of the intensity of the strongest methyl resonance (SMR), which for most proteins is observed at 0.8-0.9 ppm. The severe resonance overlap in this region facilitates detection of the SMR at low micromolar and even sub-micromolar protein concentrations. A decreased SMR intensity in the H-1 NMR spectrum of a protein mixture compared to the added SMR intensities of the isolated proteins reports that the proteins interact (SMR method). Decreased SMR intensities in ID C-13-edited H-1 NMR spectra of C-13-labeled proteins upon addition of unlabeled proteins or macromolecules also demonstrate binding (SMRC method). Analysis of the interaction between XIAP and Smac, two proteins involved in apoptosis, illustrates both methods. A study showing that phospholipids compete with the neuronal core complex for Ca2+-dependent binding to the presynaptic Ca2+-sensor synaptotagmin 1 illustrates the usefulness of the SMRC method in studying multicomponent systems.