High-Resolution Diffusion Measurements of Proteins by NMR under Near-Physiological Conditions

Measuring the translational diffusion of proteins under physiological conditions can be very informative, especially when multiple diffusing species can be distinguished. Diffusion NMR or diffusion-ordered spectroscopy (DOSY) is widely used to study molecular diffusion, where protons are used as probes, which can be further edited by the proton-attached heteronuclei to provide additional resolution. For example, the combination of the backbone amide protons (¹H^N) to measure diffusion with the well-resolved ¹H/¹⁵N correlations has afforded high-resolution DOSY experiments. However, significant amide-water proton exchange at physiological temperature and pH can affect the accuracy of diffusion data or cause complete loss of DOSY signals. Although aliphatic protons do not exchange with water protons, and thus are potential probes to measure diffusion rates, ¹H/¹³C correlations are often in spectral overlap or masked by the water signal, which hampers the use of these correlations. In this report, a method was developed that separates the nuclei used for diffusion (α protons, ¹H^α) and those used for detection (¹H/¹⁵N and ¹³C'/¹⁵N correlations). This approach enables high-resolution diffusion measurements of polypeptides in a mixture of biomolecules, thereby providing a powerful tool to investigate coexisting species under physiologically relevant conditions.