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) provides a powerful spectroscopic approach for tracking the proximity and orientation of fluorescently labeled proteins. In yeast cytosolic lysates this technique enables noninvasive monitoring of dynamic protein interactions and conformational changes under near physiological conditions. Such capability is essential for understanding cellular processes at the molecular level and for developing assays in drug discovery and quality control.

Objectives and Study Overview

The primary goal of this study was to establish and validate a FRET assay using a dual fluorescent protein fusion composed of blue fluorescent protein and green fluorescent protein linked by a protease sensitive peptide. The assay was performed in Saccharomyces cerevisiae lysates to detect energy transfer and to demonstrate loss of FRET upon enzymatic cleavage of the linker by trypsin.

Methodology and Instrumentation

Sample Preparation

• Yeast strain YRD15 expressing a BFP peptide GFP fusion with a 27 amino acid trypsin cleavage site
• Lysis performed with proprietary reagent followed by dilution in Tris buffer

Instrument Configuration

• Agilent Cary Eclipse fluorescence spectrophotometer equipped with xenon flash lamp
• Multicell Peltier holder and temperature controller set to 25 °C to optimize protease activity
• Quartz cuvettes, thermal software for scan control

Measurement Protocol

• Excitation at 360 nm to selectively excite BFP
• Emission spectral scans from 400 to 550 nm recorded at baseline and at intervals after 0.25 μg trypsin addition

Main Results and Discussion

Initial emission spectra showed a pronounced peak at 510 nm indicating efficient energy transfer from excited BFP to GFP. Following addition of trypsin and incubation at 25 °C, the green emission peak decreased progressively over 33 minutes while the blue emission peak increased correspondingly. This shift confirmed that FRET was dependent on the physical linkage of the two fluorescent proteins and that cleavage abrogated the resonance energy transfer. Instrument internal filters and flash lamp minimized background autofluorescence and photobleaching, ensuring high selectivity and sensitivity.

Benefits and Practical Applications

The described FRET assay provides a rapid and sensitive method for monitoring protein conformational changes and interactions in cell lysates. Key advantages include noninvasive detection, compatibility with standard spectrofluorometers, and real time kinetic monitoring. Applications range from fundamental studies of protein dynamics to screening of protease inhibitors and other ligands affecting protein structure.

Future Trends and Applications

Advances may include integration with live cell and high throughput screening formats, expansion to other fluorescent protein pairs, and multiplexed FRET assays for complex interaction networks. Coupling FRET with microfluidic platforms and automated data analysis will further enhance throughput and utility in pharmaceutical research and industrial process monitoring.

Conclusion

This work demonstrates that the Agilent Cary Eclipse system with Peltier temperature control can accurately track FRET changes in yeast cytosolic lysates. The approach offers a versatile platform for probing protein interactions and conformational events in a controlled environment, paving the way for broader adoption in research and QAQC laboratories.

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