尽管K-Ras蛋白在许多不同类型的人类癌症中发挥着重要作用，但在原子水平信息方面存在严重的缺口，这严重限制了我们对其在健康和疾病中的功能的理解。在这里，我们通过溶液核磁共振光谱法报告了K-Ras的定量主链结构动力学，包括与鸟苷三磷酸（GTP）核苷酸结合的野生型K-Ras活态以及两种异常表皮生长因子放射P环突变体G12D和G12C。我们使用一种新的纳米颗粒辅助的自旋松弛方法、弛豫分散和化学交换饱和转移实验覆盖了从皮秒到毫秒的完整时间尺度范围。我们结合使用这些实验，可以检测和分析功能上关键的开关I和开关II区域，这些区域以前在X射线晶体学和核磁共振光谱法中很难观察到。我们的数据揭示了从K-Ras·GTP到高度动态的激发态的协同转变，这与部分无序的K-Ras·GDP状态非常相似。这些结果提升了我们对野生型和突变体的差异GTP酶活性和信号传导特性的理解，并可能为新型治疗策略的开发提供指导。©2023. 作者。

Despite the prominent role of the K-Ras protein in many different types of human cancer, major gaps in atomic-level information severely limit our understanding of its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance spectroscopy. Our data reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited state that closely resembles the partially disordered K-Ras·GDP state. These results advance our understanding of differential GTPase activities and signaling properties of the wild type versus mutants and may thus guide new strategies for the development of therapeutics.© 2023. The Author(s).