| Method |
G protein activation rate was determined from FRET time course and dose–response data. Researchers constructed strains possessing genomic copies of the FRET pairs CFP-GPA1 (Ga) and STE18-YFP (G?), which replaced their cognate genes. They observed the dose-dependent loss of FRET on a-factor addition, which was quantitated by fluorometer. The kinetics and dose response of G protein activation were measured and compared with the pheromone responsiveness of two downstream events, cell-cycle arrest, and transcriptional activation of pheromone-inducible genes. Finally, researchers fit the data to a mathematical model that furnishes a detailed description of the yeast heterotrimeric G protein cycle (Fig. 1) and also enables quantitative explanations of the data. |