Xiang Xinyao, Hansen Alexandar L, Bruschweiler-Li Lei, Brüschweiler Rafael, Xie Mouzhe
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
Campus Chemical Instrument Center, The Ohio State University, Columbus, OH, USA.
Nat Protoc. 2025 Jul 8. doi: 10.1038/s41596-025-01177-1.
Proteins under physiological conditions have an intrinsically dynamic nature; they sample a multitude of different conformational substates that allow them to perform their biological functions. Protein motions can take place on a wide range of timescales. Although there are many different NMR experiments with sensitivity to different time windows, it has proven difficult to measure intramolecular motions that happen in the nanosecond-to-microsecond regime. Nanoparticle-assisted NMR spin relaxation (NASR) has recently been introduced to overcome this long-standing challenge. When colloidal nanoparticles are added to proteins in solution, the effective global tumbling of the protein molecules slows down, whereas the internal motions remain essentially unperturbed. NASR extends the protein dynamics observation window from picoseconds all the way into the microsecond range. In this protocol, the NASR effect is realized by using commercially available silica nanoparticles, and NMR measurements are acquired by using a standard high-field solution NMR spectrometer. NASR data analysis is shown to be straightforward. We demonstrate NASR by detecting sub-microsecond dynamics in the Switch I and II regions of oncogenic human KRAS and in the Loop I region of bacterial colicin-immunity protein Im7, among other protein constructs. When an isotope-labeled protein sample is available, this protocol can be executed in 2-5 d, including sample preparation, NMR experiments and data processing and analysis, to uncover potentially functionally important intramolecular dynamics at atomic resolution on timescales that are several orders of magnitude slower than what conventional spin relaxation experiments can observe.
在生理条件下，蛋白质具有内在的动态特性；它们会采样多种不同的构象亚态，从而能够执行其生物学功能。蛋白质运动可以在广泛的时间尺度上发生。尽管有许多不同的核磁共振（NMR）实验对不同的时间窗口敏感，但事实证明，测量在纳秒到微秒范围内发生的分子内运动是困难的。最近引入了纳米颗粒辅助的NMR自旋弛豫（NASR）来克服这一长期存在的挑战。当将胶体纳米颗粒添加到溶液中的蛋白质中时，蛋白质分子的有效整体翻滚减慢，而内部运动基本上保持不受干扰。NASR将蛋白质动力学观察窗口从皮秒一直扩展到微秒范围。在本方案中，通过使用市售的二氧化硅纳米颗粒实现NASR效应，并使用标准的高场溶液NMR光谱仪进行NMR测量。结果表明，NASR数据分析很简单。我们通过检测致癌人类KRAS的开关I和II区域以及细菌大肠菌素免疫蛋白Im7的环I区域等蛋白质构建体中的亚微秒动力学来证明NASR。当有同位素标记的蛋白质样品时，本方案可以在2-5天内完成，包括样品制备、NMR实验以及数据处理和分析，以在比传统自旋弛豫实验所能观察到的慢几个数量级的时间尺度上，以原子分辨率揭示潜在的功能重要分子内动力学。
