Genetically Encoding Quinoline Reverses Chromophore Charge and Enables Fluorescent Protein Brightening in Acidic...

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Imaging

Background:

• Acidic vesicles and organelles are crucial for cellular processes like endocytosis and degradation.
• Conventional fluorescent proteins are often nonfluorescent at acidic pH, hindering visualization of these organelles.
• Existing fluorescent reporters cause background noise, complicating the study of acidic cellular compartments.

Purpose of the Study:

• To develop a novel fluorescent protein that is specifically bright in acidic conditions.
• To overcome the limitations of existing fluorescent proteins in imaging acidic cellular environments.
• To enable spatiotemporal resolution studies of molecules within acidic vesicles and organelles.

Main Methods:

• Engineered an acid-brightening fluorescent protein (abFP) by incorporating a quinoline-containing amino acid (Qui) into the EGFP chromophore.
• Modified the chromophore's charge properties to achieve fluorescence increase at acidic pH.
• Validated abFP performance in vitro, in E. coli, and in mammalian cells, including imaging of abFP-tagged receptors in lysosomes.

Main Results:

• The novel abFP exhibits strong fluorescence at acidic pH and is nonfluorescent at neutral pH, reversing the typical pH-fluorescence profile.
• Incorporation of Qui resulted in a cationic chromophore, leading to pH-dependent fluorescence.
• abFP-tagged proteins were successfully imaged in lysosomes without background interference, unlike EGFP-tagged proteins.

Conclusions:

• The Qui-rendered cationic chromophore strategy offers a new approach for developing acid-specific fluorescent probes.
• abFPs provide a powerful tool for studying molecules associated with acidic vesicles and organelles with minimal background.
• This strategy can be extended to create a palette of colors for advanced acidic imaging across various model systems.