Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity

Area of Science:

• Biochemistry
• Cell Biology
• Spectroscopy

Background:

• HdeA is an acid-resistant chaperone known to undergo pH-dependent conformational changes.
• Monitoring intracellular pH is crucial for understanding cellular processes.
• Developing environment-sensitive probes is key for live-cell imaging.

Purpose of the Study:

• To develop a novel method for live-cell pH measurements.
• To utilize HdeA as a scaffold for an environment-sensitive fluorescent probe.
• To investigate pH-mediated conformational changes in HdeA.

Main Methods:

• Site-specific attachment of an environment-sensitive green fluorophore to the HdeA protein.
• Characterization of fluorescence changes in response to varying pH conditions.
• Live-cell imaging to monitor intracellular pH dynamics.

Main Results:

• A specific attachment site on HdeA was identified for the fluorophore.
• The attached fluorophore exhibited a significant fluorescence increase upon acidification.
• The probe successfully reported on pH changes in real-time within live cells.

Conclusions:

• The developed HdeA-fluorophore conjugate serves as a sensitive probe for live-cell pH monitoring.
• This approach allows for the study of acid-resistant chaperones and cellular pH homeostasis.
• The method provides a new tool for investigating biological processes influenced by pH gradients.