Exploiting ¹⁹F NMR in a Multiplexed Assay for Small GTPase Activity

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.