Fast hydrogen exchange affects ¹⁵N relaxation measurements in intrinsically disordered proteins

Abstract

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, ¹H–¹⁵N HSQC, is used to measure the ¹⁵N transverse relaxation rate (R₂), the measured R₂ rate is convoluted with the HX rate (k_HX) and has higher apparent R₂ values. Since the ¹⁵N R₂ measurement is important for analyzing protein backbone dynamics, the HX effect on the R₂ measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing ¹⁵N R ^CPMG₂ experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R ^CPMG₂ can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D₂O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R ^CPMG₂ values be obtained by methods described herein.