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Photoconversion in orange and red uorescent proteins
2009 Nature America, Inc. All rights reserved.
Gert-Jan Kremers1, Kristin L Hazelwood2, Christopher S Murphy2, Michael W Davidson2 & David W Piston1
We found that photoconversion is fairly common among orange and red uorescent proteins, as in a screen of 12 proteins,8 exhibited photoconversion. Specically, three red uorescent proteins could be switched to a green state, and two orange variants could be photoconverted to a far-red state. The orange proteins are ideal for dual-probe highlighter applications, and they exhibited the most red-shifted excitation of all uorescent proteins described to date.
Photoconvertible uorescent proteins (pc-FPs) exhibit a change in uorescence excitation and emission spectra after excitation at a specic wavelength and are thus useful as optical highlighters. Since the rst description of a photoactivatable probe, PA-GFP1, which switches from a dark to a uorescent state, many uorescent proteins have been developed that permit photoconversion rather than just photoactivation. These uorescent proteins switch between two colors (typically from green to red), both of which can be visualized. The palette of currently useful pc-FPs includes PS-CFP2, Dendra3,4, EosFP5, Kaede6 and KikGR7, all of which exhibit red-shifted uorescence emission accompanied by a concomitant shift in the excitation maxima after irradiation with near-UV light or deep-blue light.
The dimeric red uorescent protein Katushka and its monomeric variant mKate yield the greatest uorescence emission above 650 nm of all presently characterized red uorescent proteins8.
We found that cells expressing Katushka or mKate were uorescent upon two-photon excitation at 750 nm but that the uorescence decreased rapidly. When we subsequently imaged the cells through a GFP emission lter, we observed the appearance of green uorescence (Fig. 1a,b), indicating a photoconversion of the red uorescent proteins to a green state. Subsequent investigation revealed that red-to-green photoconversion also occurred upon single-photon laser excitation at 405 and 561 nm (Table 1) and that the green state had excitation and emission maxima of 495 and 518 nm, respectively. Katushka, mKate, mTagRFP9 and mTagRFP-T (a variant with increased photostability)10 are all derived from the
same sea anemone protein, EqFP578. All four proteins have the same chromophore (Met-Tyr-Gly), but we observed no photocon-version of mTagRFP or mTagRFP-T at any excitation wavelength.
HcRed1 is a far-red uorescent protein that had...