Abstract
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. (19)F NMR of fluorinated proteins or ligands can overcome these drawbacks. Using indole position-specific (19)F 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 (19)F-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.