Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
RIKEN Center for Biosystems Dynamics Research, Yokohama, Kanagawa, Japan.
Methods Mol Biol. 2024;2797:237-252. doi: 10.1007/978-1-0716-3822-4_17.
The activation level of RAS can be determined by GTP hydrolysis rate (k) and GDP-GTP exchange rates (k). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the k and k for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the k and k of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the k and k simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.
RAS 的激活水平可以通过 GTP 水解速率 (k) 和 GDP-GTP 交换速率 (k) 来确定。无论是 GTP 水解受损还是 GDP-GTP 交换增强，都会导致致癌突变体中 RAS 的异常激活。因此，定量测定 k 和 k 对于理解 RAS 致癌机制和药物开发非常重要。传统方法分别测量了 RAS 的 k 和 k。然而，在细胞内环境中，在保持 GTP 浓度不变的条件下，GTP 水解和 GDP-GTP 交换反应同时发生。此外，RAS 的细胞内活性受到内源性调节蛋白的影响，如 RAS GTP 酶激活蛋白 (GAPs) 和鸟嘌呤核苷酸交换因子 (GEFs)。在这里，我们描述了体外和细胞内 NMR 方法，通过在恒定 GTP 浓度条件下测量 GTP 结合比的时变来同时估计 k 和 k。
