Fluorescent BODIPY-GTP analogs: Real-time measurement of nucleotide binding to G proteins

Three BODIPY GTP gammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BOD IPY FL GTP molecules were synthesized as possible fluorescent probes to stu dy guanine nucleotide binding spectroscopically. Binding to G(alpha omicron ) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-f old, respectively. Binding of GTP gammaS and GppNHp analogs to G(alpha omic ron) is of high affinity (K-D, 11, 17, 55, and 110 nM, respectively) and re aches a stable plateau while fluorescence of BODIPY FL GTP shows a transien t increase which returns to baseline. Furthermore, BODIPY FL GTP gammaS sho ws varying affinities for alpha (omicron), alpha (s), alpha (i1), and alpha (i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTP gammaS. Hal f-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(a lpha) subunit is most likely due to a rotation around the y-thiol (GTP gamm aS) or the 3 ' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BO DIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODI PY moiety to the external environment, as seen by an increase in sodium iod ide quenching. The visible excitation and emission spectra and high fluores cence levels of these probes permit robust real-time detection of nucleotid e binding. (C) 2001 Academic Press.