N transverse relaxation measurements for the characterization of µs-ms dynamics are deteriorated by the deuterium isotope effect on N resulting from solvent exchange.

N R relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide-water exchange can severely skew Hahn-echo based N R relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent N R rate constants-which should not be mistaken with protein inherent chemical exchange contributions, R, to N R. For measurements of N R rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low DO molar fraction in the sample buffer, as low as 1%, or the use of an external DO reference along with a modified N R Hahn-echo based experiment. This combination allows for the measurement of R contributions to N R originating from conformational exchange in a time window from µs to ms.