Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

Abstract

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.

Keywords: :

• binding assay
• fluorescent protein
• intermolecular FRET
• large Stokes shift

Author contributions

TN conceived the project. CSC performed most of the experiments. FR and ILG determined Kd value between EPAC1_CNBD-mAmetrine and 8-[DY-547]-AET-cAMP. CSC and TN wrote the manuscript. CSC and ILG prepared the figures. All authors revised and approved the manuscript.

Acknowledgments

We thank Dr Robert Campbell for providing pBAD-mAmetrine1.2. We appreciate the National Institute of Genetics for providing two E. coli strains, JM109 (DE3) and BL21 (DE3). We thank Eugenio López, Jorge Yáñez and Paul Gaytán for oligonucleotide synthesis and plasmid sequencing. We also thank Dr. Alejandra Covarrubias for sharing the FS5 Spectrofluorometer with SC-40.

Financial competing interests disclosure

This work was supported by the following grants: CONACYT-Ciencia Básica 177138 (TN), 128566 (Consorcio de fisiología del espermatozoide), CONACYT-Fronteras de la ciencia 71, UNAM/DGAPA-PAPIIT IN203513 (TN), IN206116 (TN), IN205518 (ILG). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2144/btn-2018-0041

Additional information

Funding

This work was supported by the following grants: CONACYT-Ciencia Básica 177138 (TN), 128566 (Consorcio de fisiología del espermatozoide), CONACYT-Fronteras de la ciencia 71, UNAM/DGAPA-PAPIIT IN203513 (TN), IN206116 (TN), IN205518 (ILG). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed