Photoswitchable cyan fluorescent protein as a FRET donor.
Among a variety of fluorescent proteins available today, there is a lack of suitable markers with excitation/emission in the violet/blue part of visible spectrum. Recently, we reported on photoswitchable cyan fluorescent protein (PS-CFP), which represents monomeric high-contrasting photactivatable label for in vivo protein movement tracking. However, PS-CFP demands high intensity of light for the photoswitching. Therefore it can be employed as a common fluorescent tag at conventional light intensities, which cause negligible or zero photoactivation. High pH stability and unique positioning of excitation/emission peaks make it a worthy supplement to the existing palette of fluorescent proteins. Here we use PS-CFP fusion with a yellow fluorescent protein phiYFP to show that PS-CFP is a promising donor partner for the fluorescence resonance energy transfer (FRET). A remarkable phenomenon is that PS-CFP donor fluorescence turned to be essentially stable with and without FRET, while acceptor emission demonstrated record dynamic range of up to 7.8-fold. This makes the FRET pair presented a useful tool for the single color high throughput screenings. Here we also propose ways for further PS-CFP enhancing, aiming to develop bright cyan fluorescent protein with unique spectral characteristics.
Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Ekaterina A
EA
Souslova
Dmitriy M
DM
Chudakov
D001426:Bacterial Proteins / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000528:radiation effects*; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins