H/D exchange of a ¹⁵N labelled Tau fragment as measured by a simple Relax-EXSY experiment

We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T₁ relaxation rates in solvents of different D₂O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.