Exploiting <sup>19</sup>F NMR in a Multiplexed Assay\nfor Small GTPase Activity

Small GTPases (smG)\nare a 150-member family of proteins, comprising\nfive subfamilies: Ras, Rho, Arf, Rab, and Ran-GTPases. These proteins\nfunction as molecular switches, toggling between two distinct nucleotide-bound\nstates. Using traditional multidimensional heteronuclear NMR, even\nfor single smGs, numerous experiments, high protein concentrations,\nexpensive isotope labeling, and long analysis times are necessary. <sup>19</sup>F NMR of fluorinated proteins or ligands can overcome these\ndrawbacks. Using indole position-specific <sup>19</sup>F labeling\nof the proteins, the activities of several smGs were measured in a\nmultiplexed fashion. We investigated 4-, 5-, 6-, and 7-fluoro tryptophan\ncontaining smGs to study nucleotide binding. Distinct resonances for\nGDP- or GTP-bound states of three different <sup>19</sup>F-labeled\nsmGs, RhoA, K-Ras, and Rac1, were observed, and the kinetics of exchange\nand hydrolysis were measured. This multiplexed system will permit\nscreening of nucleotide-specific ligands of smGs under true physiological\nconditions.