Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements.

Abstract Protein–protein interactions are necessary for various cellular processes, and therefore, information related to protein–protein interactions and structural information of complexes is invaluable. To identify protein–protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein–protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an 15 N-labeled amino acid and several amino acids 13 C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω 1 -TROSY-type backbone 1 H– 15 N and aromatic 1 H– 13 C shift correlations together with single quantum methyl 1 H– 13 C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein–protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein–protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein–protein complex, for instance, as the first step in structure-guided drug development.