Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.
J Biomol NMR. 2013 Mar;55(3):249-56. doi: 10.1007/s10858-013-9706-1. Epub 2013 Jan 12.
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, (1)H-(15)N HSQC, is used to measure the (15)N transverse relaxation rate (R2), the measured R2 rate is convoluted with the HX rate (kHX) and has higher apparent R2 values. Since the (15)N 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 (15)N R 2 (CPMG) 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 2 (CPMG) 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 2 (CPMG) values be obtained by methods described herein.
未保护酰胺质子可与溶剂质子发生快速氢交换 (HX)。一般来说，使用酰胺质子检测的往返相干转移的 NMR 实验受到快速 HX 的影响，导致信号强度降低。当这些实验之一，（1）H-（15）N HSQC，用于测量（15）N 横向弛豫率（R2）时，测量的 R2 率与 HX 率（kHX）卷积，并具有更高的表观 R2 值。由于（15）N R2 测量对于分析蛋白质骨架动力学很重要，因此在此通过多指数信号衰减来研究和描述 HX 对 R2 测量的影响。我们通过在暴露于溶剂的α-突触核蛋白（一种固有无序蛋白）上执行（15）N R2（CPMG）实验来证明这些效应。我们表明，可以通过导出的方程提取 R2（CPMG）上的 HX 效应。总之，HX 效应可能是脉冲序列特异性的，源自各种来源，包括 J 耦合演化、稳态水质子磁化的变化以及样品中的 D2O 含量。为避免 HX 对未保护酰胺的弛豫数据分析的影响，建议考虑对 HX 不敏感的 NMR 实验条件，或者通过本文所述的方法获得固有 R2（CPMG）值。
