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.

Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3

“…29 Actually, GFP has a relatively high pKa value, 6.0, 29 whereas, the pKa value of mRFP is 4.5. 19 We therefore investigated the effect of the acidic conditions of the lysosome on fluorescence.…”

“…Attenuation of fluorescence is caused by protonation of its fluorophore and could occur within <1 ms, whereas protein conformational changes or degradation could occur over millisecond and longer time frames. 29 Therefore, we can monitor the maturation process only for a short time after fusion takes place.…”

The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

“…This indicates that the soluble GFP has been secreted into the apoplast and released into the culture medium. The absorbance and fluorescence properties of GFPS65T are very sensitive to pH, and its fluorescence is reported to be completely suppressed below pH 6.0 (Haupts et al, 1998;Kneen et al, 1998). Thus, pH sensitivity may be one explanation for the quenching observed when GFP was released into the apoplast of Arabidopsis plants or the tobacco cell culture medium, both of which are acidic.…”

Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators

“…[22][23][24]. GFP is most often used as a passive indicator of gene expression and protein localization, but variants have been constructed that act as indicators of intracellular concentrations of H ϩ , Ca 2ϩ , and halide ions (25)(26)(27)(28)(29)(30)(31)(32). The key properties of GFP are efficient fluorescence, protease resistance, and stability throughout a wide range of pH and solvent conditions.…”