Abstract
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 interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.
Highlights
Knowledge of protein−protein interactions is essential for the understanding of many biological processes
We introduced 1H and 15N solvent Paramagnetic relaxation enhancement (PRE) as a general and powerful tool for characterizing intermolecular interfaces in large biomolecular complexes in the solid state
We demonstrate for 100% backexchanged protein in the presence of a fully protonated binding partner that 1H−1H spin diffusion is sufficiently slowed down at 100 kHz to allow the use of 1H R1 as a site-specific probe of solvent accessibility. 1H R1 and 15N R1 are the most sensitive probes, enabling accurate measurement of even small PREs
Summary
Knowledge of protein−protein interactions is essential for the understanding of many biological processes. We previously observed large chemical shift perturbations (CSPs) for T53, V54, and L7 upon GB1:IgG complex formation but were unable to completely explain their origin (Figure 5a; L7 could potentially be explained by a small backbone conformation change indicated by change in Cα secondary chemical shift of L6Cα compared to solution data). The presence of these CSPs and the elevated ΔsPREs suggest that there might be an additional interaction between GB1 and IgG, which involves β4 and is not observed in the complexes of protein G domains with IgG fragments. The local environment of GB1 in the complex would be defined almost entirely by the specific interactions with IgG, leaving GB1 largely unaffected by any heterogeneity of the sample
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