Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we review our recently developed methods that greatly simplify PPI studies, which minimize the number of samples required to fully characterize residues involved in the protein-protein binding interface. This original strategy combines asymmetric labeling of two binding partners and the carbonyl-carbon label selective (CCLS) pulse sequence element implemented into the heteronuclear single quantum correlation (¹H-15N HSQC) spectra. The CCLS scheme removes signals of the J-coupled 15N⁻13C resonances and records simultaneously two individual amide fingerprints for each binding partner. We show the application to the measurements of chemical shift correlations, residual dipolar couplings (RDCs), and paramagnetic relaxation enhancements (PRE). These experiments open an avenue for further modifications of existing experiments facilitating the NMR analysis of PPIs.
Keywords: Carbonyl Carbon Label Selective (CCLS); dual carbon label selective (DCLS); nuclear magnetic resonance (NMR); paramagnetic relaxation enhancement (PRE); protein-protein interactions (PPI); residual dipolar coupling (RDC).