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15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

  • Pratibha Kumari
  • Lukas Frey
  • Alexander Sobol
  • Nils-Alexander Lakomek
  • Roland Riek
Article
  • 153 Downloads

Abstract

15N R2 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 15N R2 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 15N 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 15N R2 rate constants—which should not be mistaken with protein inherent chemical exchange contributions, Rex, to 15N R2. For measurements of 15N R2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D2O molar fraction in the sample buffer, as low as 1%, or the use of an external D2O reference along with a modified 15N R2 Hahn-echo based experiment. This combination allows for the measurement of Rex contributions to 15N R2 originating from conformational exchange in a time window from µs to ms.

Keywords

Intrinsically disordered proteins NMR relaxation experiments Amide exchange Deuterium isotope effect Loop dynamics 

Supplementary material

10858_2018_211_MOESM1_ESM.pdf (409 kb)
Supplementary material 1 (PDF 409 KB)

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© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratory of Physical Chemistry, Department of Chemistry and Applied BiosciencesETH ZürichZurichSwitzerland

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