Identification and Characterisation of a pH-stable GFP

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Green fluorescent proteins (GFPs) are essential tools in cell biology for visualizing cellular processes.
• Existing GFPs often lose brightness and stability in acidic environments, limiting their use in quantitative studies under such conditions.
• There is a need for a GFP variant that maintains fluorescence in low-pH cellular compartments.

Purpose of the Study:

• To engineer and characterize a novel GFP variant with retained brightness and stability at low pH.
• To develop a reliable fluorescent marker for quantitative analysis of acidic cellular environments.
• To enable precise monitoring of cellular processes occurring in low-pH conditions.

Main Methods:

• Engineering of a tandem dimer GFP (tdGFP) construct.
• Characterization of the engineered pH-tdGFP's fluorescence properties across a range of pH values.
• Assessment of pH-tdGFP stability and brightness under acidic conditions.

Main Results:

• The engineered pH-tdGFP demonstrates significant brightness and stability in acidic conditions.
• pH-tdGFP maintains efficient excitation and fluorescence properties even at low pH.
• The novel GFP variant overcomes the limitations of conventional GFPs in acidic environments.

Conclusions:

• pH-tandem dimer GFP (pH-tdGFP) is a robust fluorescent protein suitable for low-pH environments.
• This engineered GFP can serve as a quantitative marker for studying processes like endocytosis, autophagy, and starvation.
• pH-tdGFP offers a valuable tool for advancing quantitative cell biology research in acidic conditions.