Departments of Molecular Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research, Hyderabad, Telangana, 500107, India.
Angew Chem Int Ed Engl. 2019 May 6;58(19):6250-6254. doi: 10.1002/anie.201900241. Epub 2019 Apr 4.
Molecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl H CPMG-based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY-effect to increase signal-to-noise. Complexities from the multiplicity of methyl H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.
分子复合物通常会采样能够指导其特定功能的构象状态。这些状态可能很难通过传统的生物物理方法来观察，但可以使用多种不同的 NMR 自旋弛豫实验来研究。然而，当这些应用聚焦于中等至高分子量的蛋白质时，由于快速弛豫的信号会降低光谱的灵敏度和分辨率，因此变得复杂。这里描述了一种基于甲基 H CPMG 的实验，用于研究蛋白质机器的激发构象状态，该实验利用 TROSY 效应来提高信号噪声比。通过处理甲基 H 跃迁的多重性来解决复杂性问题，以生成一种稳健的脉冲方案，并将其应用于 320 kDa 的平衡蛋白 p97。
