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 (Rg) obtained from small-angle x-ray scattering (SAXS) experiments or the hydrodynamic radius (Rh) 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 Rhusing 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 Rhis therefore important. We have revisited the basis for the commonly used reference value for the Rhof 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 Rhreference value for dioxane Rhis 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 Rhvalues for proteins whose hydrodynamic radii have been measured by PFG NMR. These results are particularly important when the absolute value of Rhis of interest such as when determining or validating ensemble descriptions of intrinsically disordered proteins.