title: Green fluorescent protein as a noninvasive intracellular pH indicator.
abstract: It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.
authors.affiliation: Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco 94143-0521, USA.
authors.forename: M
authors.initials: M
authors.lastname: Kneen
authors.forename: J
authors.initials: J
authors.lastname: Farinas
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers; D016466:CHO Cells; D002138:Calibration; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D015415:Biomarkers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000643:Ammonium Chloride; D049452:Green Fluorescent Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone