NMR Methodologies for the Analysis of Protein-Protein Interactions

This chapter reviews current state-of-the-art NMR approaches for studying the structure and dynamics of protein complexes. Practical aspects and the workflow for such studies are presented. Protocols, tips and tricks, as well as troubleshooting of experiments are discussed. The most efficient approach for determining the quaternary structure of multidomain proteins and complexes starts from the structures of individual domains and subunits. The arrangement of the domains/subunits within the complex is then defined by NMR-derived information about the domain interfaces, combined with (long-range) distance and orientational restraints. The following aspects of the structural analysis of protein complexes are discussed: (i) sample preparation and isotope labeling; (ii) NMR tools for determining binding interfaces: chemical shift perturbations, hydrogen exchange rates, nuclear Overhauser effects, isotope editing/filtering, cross-saturation, and differential line broadening; and (iii) defining the domain/subunit arrangement using residual dipolar couplings, pseudocontact shifts, paramagnetic relaxation enhancements, and NMR relaxation. The applications of these state-of-the-art NMR techniques are illustrated with studies of challenging (high-molecular-weight) protein complexes.