Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin

Genetically encoded pH sensors based on fluorescent proteinsarevaluable tools for the imaging of cellular events that are associatedwith pH changes, such as exocytosis and endocytosis. SupereclipticpHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variantwidely used for such applications. Here, we report the rational design,development, structure, and applications of Lime, an improved SEPvariant with higher fluorescence brightness and greater pH sensitivity.The X-ray crystal structure of Lime supports the mechanistic rationalethat guided the introduction of beneficial mutations. Lime providessubstantial improvements relative to SEP for imaging of endocytosisand exocytosis. Furthermore, Lime and its variants are advantageousfor a broader range of applications including the detection of synapticrelease and neuronal voltage changes.

Automated summary

This article introduces genetically encoded pH sensors based on fluorescent proteins for imaging cellular events associated with pH changes. A new improved variant called Lime with higher fluorescence brightness and greater pH sensitivity is presented. Lime shows significant improvements over SEP in endocytosis and exocytosis imaging, and is advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.