Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of α-spectrin by MAS solid-state NMR

Water molecules are a major determinant of protein stability and are important for understanding protein-protein interactions. We present two experiments which allow to measure first the effective T₂ decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H₂O to H^N using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly ²H, ¹⁵N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H₂O-H^N distances which were extracted from the X-ray structure of the protein. We find that the H^N T₂ decay rates and H₂O-H^N build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton.