<abstract xmlns="http://www.w3.org/1999/xhtml"><p>The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRET<sub>eff</sub> values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.</p></abstract>

The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.

Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational...

Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

Published Online: Aug 22, 2018

Page range: 252 - 258

Received: Feb 28, 2017

Accepted: Mar 23, 2018

DOI: https://doi.org/10.2478/prolas-2018-0065

Keywords
Calcium Release-Activated Channel (CRAC), Förster Resonance Energy Transfer (FRET), HEK 293, YFP, CFP

© 2018 Laura Hippe et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

Published Online: Aug 22, 2018

Page range: 252 - 258

Received: Feb 28, 2017

Accepted: Mar 23, 2018

DOI: https://doi.org/10.2478/prolas-2018-0065

KeywordsCalcium Release-Activated Channel (CRAC), Förster Resonance Energy Transfer (FRET), HEK 293, YFP, CFP

© 2018 Laura Hippe et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Keywords
Calcium Release-Activated Channel (CRAC), Förster Resonance Energy Transfer (FRET), HEK 293, YFP, CFP