On the use of dioxane as reference for determination of the hydrodynamic radius by NMR spectroscopy

Measuring the compaction of a protein or complex is key to our understanding of the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (R_g) obtained from small-angle x-ray scattering (SAXS) experiments or the hydrodynamic radius (R_h) obtained, for example, by pulsed field gradient NMR (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate R_h using an internal standard to account for sample viscosity and uncertainty about the gradient strength. 1,4-Dioxane is one such commonly used internal standard, and the reference value of R_h is therefore important. We have revisited the basis for the commonly used reference value for the R_h of dioxane (2.12 Å) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We followed the same approach that was used to establish the current reference value by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current R_h reference value for dioxane R_h is underestimated, and we instead suggest a new value of 2.27 ± 0.04 Å. Using this updated reference value results in a ∼7% increase in R_h values for proteins whose hydrodynamic radii have been measured by PFG NMR. These results are particularly important when the absolute value of R_h is of interest such as when determining or validating ensemble descriptions of intrinsically disordered proteins.