Characterizationof Protein–Protein Interfacesin Large Complexes by Solid-State NMR Solvent Paramagnetic RelaxationEnhancements

摘要

Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein–protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein–protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that ¹H and ¹⁵N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein–protein interfacesin the GB1 complex with immunoglobulin G (IgG). Our results suggestthe potential existence of an additional binding site and providenew insights into GB1:IgG complex structure that amend and revisethe current model available from studies with IgG fragments. We demonstratesPREs as a practical, widely applicable, robust, and very sensitivetechnique for determining intermolecular interaction interfaces inlarge biomolecular complexes in the solid state.