pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein

Green florescent protein (GFP) variants that are sen-sitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiomet-ric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidi-fication, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalian-ized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher flores-cence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and dis-plays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2 displays enhanced florescence while sustaining ratiometric pH-sensitivity, representing a significant improvement for this methodological ap-proach.