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

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Imaging

Background:

• Genetically encoded pH sensors are crucial for studying cellular processes like exocytosis and endocytosis.
• Superecliptic pHluorin (SEP) is a widely used green fluorescent protein (GFP) variant for pH imaging.
• Existing sensors have limitations in brightness and sensitivity.

Purpose of the Study:

• To rationally design and develop an improved pH-sensitive fluorescent protein variant.
• To enhance fluorescence brightness and pH sensitivity compared to existing SEP variants.
• To characterize the structure and applications of the new variant, named Lime.

Main Methods:

• Rational design principles guided the introduction of beneficial mutations into SEP.
• X-ray crystallography was used to determine the structure of the Lime protein.
• Performance of Lime was evaluated for imaging cellular events like endocytosis and exocytosis.

Main Results:

• Lime exhibits significantly higher fluorescence brightness and greater pH sensitivity than SEP.
• The crystal structure of Lime validates the design strategy and mechanistic understanding.
• Lime demonstrates superior performance in imaging endocytosis and exocytosis.
• Lime and its variants show utility in detecting synaptic release and neuronal voltage changes.

Conclusions:

• Lime represents a significant advancement in genetically encoded pH sensors.
• The improved properties of Lime enable more effective imaging of cellular pH dynamics.
• Lime and its variants offer versatile applications in neuroscience and cell biology research.