Structural and spectral response of green fluorescent protein variants to changes in pH

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria ha s become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracell ular pH, To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pat tern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (SG5G/V68L/S72A/T203Y) to change the solvent accessibi lity (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore . pH titrations of these variants indicate that the chromophore pK(a) can b e modulated over a broad range from 6 to 8, allowing for pH determination f rom pH 5 to pH 9, Finally, mutagenesis was used to raise the pK(a) from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbest ic point. This raises the interesting possibility of using fluorescence at this isosbestic point, as an internal reference. Practical real time in viv o applications in cell and developmental biology are proposed.