Monitoring fluorescence resonance energy transfer (FRET) between GFP fusions in lysates of the yeast Saccharomyces cerevisiae using the Agilent Cary Eclipse

Importance of the Topic

Fluorescence resonance energy transfer (FRET) between fluorescent proteins offers a sensitive, non‐destructive means to monitor molecular proximity and conformational changes in real time. In yeast systems, FRET using blue and green fluorescent protein variants enables dynamic studies of protein–protein interactions and ligand‐induced structural transitions, providing critical insights in cell biology and biochemistry.

Study Objectives and Overview

This application study aimed to establish a robust assay for detecting changes in FRET efficiency between a BFP–GFP fusion protein in cytosolic lysates of Saccharomyces cerevisiae, using the Agilent Cary Eclipse fluorescence spectrophotometer. By introducing a protease‐cleavable linker between the fluorophores, the method allows direct monitoring of energy transfer decay upon enzymatic cleavage.

Methodology and Instrumentation

Yeast cells expressing a gene cassette encoding BFP linked via a 27‐amino‐acid trypsin‐sensitive peptide to GFP were harvested, washed, and lysed with Y-PER reagent. Lysates were diluted in Tris/HCl buffer (pH 8.0) and transferred to 10 mm quartz cuvettes in a multicell Peltier holder maintained at 25 °C. BFP was excited at 360 nm, and emission scans were recorded from 400–550 nm before and after adding 0.25 µg trypsin at defined time intervals.

• Fluorescence Spectrophotometer: Agilent Cary Eclipse with xenon flash lamp
• Multicell Peltier holder and temperature controller
• Quartz 10 mm cuvettes (Sarstedt)
• Eclipse Thermal Software (Agilent BioMelt package)

Results and Discussion

Initial emission spectra showed a characteristic BFP peak at 450 nm and a prominent GFP peak at 510 nm, indicating efficient FRET. After trypsin addition, the GFP emission peak gradually diminished over 33 minutes, while BFP emission increased slightly, confirming cleavage of the peptide linker and loss of energy transfer. These data demonstrate that the system can sensitively detect alterations in FRET arising from proteolytic separation of donor and acceptor fluorophores in yeast lysates.

Benefits and Practical Applications

This assay provides a straightforward platform for quantitative analysis of protein–protein interactions and conformational dynamics in yeast models. The use of cytosolic lysates avoids cellular autofluorescence and enables controlled protease access, while the Cary Eclipse’s internal monochromators and temperature control minimize photobleaching and enhance signal specificity. Potential applications include drug screening, protein engineering, and studies of signaling pathways.

Future Trends and Opportunities

Advances in fluorescent protein engineering and time‐resolved detection promise improved FRET sensitivity and multiplexing capabilities. Integration with microfluidic platforms and high‐throughput screening systems could extend this approach to large‐scale interaction studies. Additionally, expanding the palette of donor–acceptor pairs may enable simultaneous monitoring of multiple interactions within complex biological networks.

Conclusion

The Agilent Cary Eclipse system coupled with a protease‐sensitive BFP–GFP fusion offers a reliable, reproducible assay for monitoring FRET changes in yeast lysates. This method facilitates detailed investigation of protein interactions and conformational shifts under controlled conditions, supporting diverse research and quality control applications in analytical biochemistry.

Reference

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