Small GTPases (smG) are a 150-member family of proteins, comprising five subfamilies: Ras, Rho, Arf, Rab, and Ran-GTPases. These proteins function as molecular switches, toggling between two distinct nucleotide-bound states. Using traditional multidimensional heteronuclear NMR, even for single smGs, numerous experiments, high protein concentrations, expensive isotope labeling, and long analysis times are necessary. ¹⁹F NMR of fluorinated proteins or ligands can overcome these drawbacks. Using indole position-specific ¹⁹F labeling of the proteins, the activities of several smGs were measured in a multiplexed fashion. We investigated 4-, 5-, 6-, and 7-fluoro tryptophan containing smGs to study nucleotide binding. Distinct resonances for GDP- or GTP-bound states of three different ¹⁹F-labeled smGs, RhoA, K-Ras, and Rac1, were observed, and the kinetics of exchange and hydrolysis were measured. This multiplexed system will permit screening of nucleotide-specific ligands of smGs under true physiological conditions.

中文翻译：

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在多重检测中利用 19F NMR 检测小 GTP 酶活性


小 GTP 酶 （smG） 是一个由 150 个成员组成的蛋白质家族，由五个亚家族组成：Ras、Rho、Arf、Rab 和 Ran-GTP 酶。这些蛋白质起着分子开关的作用，在两种不同的核苷酸结合状态之间切换。使用传统的多维异核 NMR，即使对于单个 smG，也需要进行大量实验、高蛋白质浓度、昂贵的同位素标记和较长的分析时间。¹⁹氟化蛋白质或配体的 F NMR 可以克服这些缺点。使用蛋白质的吲哚位置特异性 ¹⁹F 标记，以多重方式测量几种 smG 的活性。我们研究了含有 smGs 的 4-、5-、6-和 7-氟色氨酸，以研究核苷酸结合。观察到三种不同的 ¹⁹F 标记的 smGs RhoA、K-Ras 和 Rac1 的 GDP 或 GTP 结合状态的不同共振，并测量了交换和水解动力学。该多重系统将允许在真实生理条件下筛选 smG 的核苷酸特异性配体。