Minute Additions of DMSO Affect Protein Dynamics Measurements by NMR Relaxation Experiments through Significant Changes in Solvent Viscosity

Studies of protein−ligand binding often rely on dissolving the ligand in dimethyl sulfoxide (DMSO) to achieve sufficient solubility, and then titrating the ligand solution into the protein solution. As a result, the final protein−ligand solution contains small amounts of DMSO in the buffer. Here we report how the addition of DMSO impacts studies of protein conformational dynamics. We used (15)N NMR relaxation to compare the rotational diffusion correlation time (τ (C)) of proteins in aqueous buffer with and without DMSO. We found that τ (C) scales with the viscosity of the water−DMSO mixture, which depends sensitively on the amount of DMSO and varies by a factor of 2 across the relevant concentration range. NMR relaxation studies of side chains dynamics are commonly interpreted using τ (C) as a fixed parameter, obtained from backbone (15)N relaxation data acquired on a separate sample. Model‐free calculations show that errors in τ (C), arising from mismatched DMSO concentration between samples, lead to significant errors in order parameters. Our results highlight the importance of determining τ (C) for each sample or carefully matching the DMSO concentrations between samples.