Practical considerations for investigation of protein conformational dynamics by 15N R 1ρ relaxation dispersion.
Practical considerations for investigation of protein conformational dynamics by 15N R 1ρ relaxation dispersion.
复制标题
通过 15N R 1ρ 弛豫分散研究蛋白质构象动力学的实际考虑。
DOI：
10.1007/s10858-017-0097-6
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Sugase K.
中科院分区：
文献类型：
--
作者：
Walinda E;Morimoto D;Shirakawa M;Sugase K.
越来越明显的是，蛋白质不是静态的实体，它们的功能往往严重依赖于水溶液中多种构象状态的准确采样。因此，研究蛋白质基态结构（“激发态”）以外的构象状态的方法的发展具有至关重要的生物物理意义。在这里，我们研究的实验方案，最佳探测化学交换过程中的蛋白质在微米至毫秒的时间尺度上的15 NR 1 ρ弛豫色散。该方案使用选择性Hartmann-Hahn交叉极化（CP）转移用于激发，并且从1D NMR光谱导出峰积分（Korzhnev等人，J Am Chem Soc 127：713-721，2005;汉森等人，J Am Chem Soc 131：3818-3819，2009）。模拟和实验共同表明，在这样的CP为基础的计划，必须采取措施，以实现准确的抑制不希望的非共振相干，当使用弱自旋锁定字段。这然后（i）确保在没有化学交换的情况下的弛豫色散曲线是平坦的，并且（ii）促进在光谱的拥挤区域中的弛豫色散曲线的提取。通过以交错方式记录自由感应衰减并包括加热补偿元件，进一步提高了实验数据的质量。所报道的考虑将特别有利于使用CP-basedR 1 ρ弛豫色散来分析较大蛋白质中的构象交换过程，其中共振线重叠成为主要限制因素。
It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by15NR1ρrelaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-basedR1ρrelaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.