Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts

► NMR is used to directly monitor activation state of GTPase proteins in real time. ► Application to intrinsic nucleotide hydrolysis/exchange as well as GAP/GEF assays. ► Unambiguous readout enables assays of GAP/GEF activities in mammalian cell lysates. ► Small GTPases Ras, RhoA, and Rheb are presented examples. ► Activities of TSC2, p120 RasGAP, Sos, PDZ-RhoGEF and Lfc/GEF-H1 were assayed.