Development of a Novel GFP-based Ratiometric Excitation and Emission pH Indicator for Intracellular Studies

ABSTRACT

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E^sup 2^GFP) as an effective ratiometric pH indicator for intracellular studies. E^sup 2^GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E^sup 2^GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E^sup 2^GFP to determine the average intracellular pH (pH,) and spatial pH^sub i^ maps in two …