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 Rh 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 Rh is therefore important. We have revisited the basis for the commonly used reference value for the Rh 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 Rh reference value for dioxane Rh 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 Rh values for proteins whose hydrodynamic radii have been measured by PFG NMR. These results are particularly important when the absolute value of Rh is of interest such as when determining or validating ensemble descriptions of intrinsically disordered proteins.

中文翻译：

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关于使用二恶烷作为参考通过 NMR 波谱法测定流体动力学半径


测量蛋白质或复合物的压缩度是我们了解生物分子内部和之间相互作用的关键。在实验中，通常通过估计从小角 X 射线散射 （SAXS） 实验获得的回转半径 （Rg） 或例如通过脉冲场梯度 NMR （PFG NMR） 光谱获得的流体动力学半径 （Rh） 来探测蛋白质压缩。PFG NMR 实验通常报告平移扩散系数，而该系数又可用于使用内标估计 Rh，以考虑样品粘度和梯度强度的不确定性。1,4-二恶烷就是这样一种常用的内标，因此 Rh 的参考值很重要。我们重新审视了二恶烷 Rh （2.12 Å） 的常用参考值的基础，该值用于将测得的扩散系数转换为流体动力学半径。我们遵循了与确定当前参考值相同的方法，通过测量一组七种不同蛋白质的 SAXS 和 PFG NMR 数据并将其用作标准品。我们的分析表明，目前二氧六环 Rh 的 Rh 参考值被低估了，我们建议将新值为 2.27 ± 0.04 Å。使用这个更新的参考值会导致通过 PFG NMR 测量其流体动力学半径的蛋白质的 Rh 值增加 ∼7%。当 Rh 的绝对值值得关注时，例如在确定或验证固有无序蛋白质的集合描述时，这些结果尤为重要。