New GFPs As Donor/acceptor Pairs For Fluorescence Resonance Energy Transfer

With the development of biology, the mechanism of life phenomena especially the protein-protein interactions, become more and more important. Fluorescence resonance energy transfer(FRET) which based on green fluorescent protein (GFP) is an important method to detect the interactions of proteins. As a pair of donor/acceptor for FRET requires the donor be fluorescent and of sufficiently long lifetime, the donor molecule's fluorescence emission spectrum overlaps(to some extent) the excitation spectrum of the acceptor molecule, but few corss-talk of the two molecule's emission spectrum, high signals to background in specifically cell and have appropriate photostability.Among the genetically encoded fluorophores, two GFP color mutants—CFP and YFP have emerged as the leading donor/acceptor pair for FRET experiments.The major disadvantages of using CFP in FRET experiments are CFP's dim fluorescence, which results in a low signal-to-noise ratio, and CFP's excitation by expensive UV lasers, which hinder FRET imaging on many confocal microscopes. In this paper, we used three of the new GFP variants,named MiCy , citrine and mKO as a novel FRET pairs to overcome these disadvantages. The new GFP variants were purified with genetic engineering method,and their excitation and emission spectra were determined. The high fluorescence quantum of new GFPs, the good spectral overlap between Micy emission and citrine absorption, citrine emission and mKO absorption creates a Forster's distance for the Micy/citrine pair as large as 5.8 nm and citrine/mKO pair 5.65nm, while the value for CFP/YFP is 4.9 nm. Also, the max excitation wavelength of Micy is more fit to Ar laser 457nm line and Citrine, mKO exhibits a less pH-sensitivity and photobleach. By using Ni-NTA-agarose as FRET medol, FRET between MiCy/citrine pair, citrine/mKO were successfully detected no matter on fluorescence spectrophotometer or on confocal microscope.Firstly, MiCy, Citrine, mKO were inser into expression vectors of pRSETB, and then MiCy, Citrine,mKO were expressed in E.coli and purified by using Ni-NTA collum chromatography.Secondly, we investigated the spectroscopy character of the three GFPs. We calculated the Forster distance of the MiCy/Citrine pair and the Citrine/mKO pair. Thereby, we exploredthe application of Micy-Citrine as a new FRET donor-acceptor pair. The results indicated that no matter on Fluorescence Spectroscopy or on Confocal all can measure the FRET phenomenons.Finally, we explored the application of MiCy-Citrine-mKO as a triple FRET pair. The FRET phenomenon of the triple FRET pair can obviously be observed on Fluorescence Spectroscopy.