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Microscopy Reference Library

Anthozoa (Coral) Fluorescent Proteins

The search for a red-emitting fluorescent protein with performance attributes similar to those of the enhanced green fluorescent protein (EGFP) from the Aequorea victoria jellyfish (in effect, brightness, photostability, and utility in fusions) has been seen as a critical avenue to providing an important tool for multicolor imaging and in generating new fluorescence resonance energy transfer (FRET) biosensors with spectral profiles in the longer wavelength regions. To address this problem, several groups of investigators demonstrated that much of the color diversity in reef corals is a result of GFP-like proteins. It is widely thought that these proteins evolved in the corals to fulfill roles that are distinct from those in the luminescent jellyfish.

Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., and Lukyanov, S. A.

Fluorescent proteins from nonbioluminescent Anthozoa species.  Nature Biotechnology 17: 969-973 (1999).  The first report of fluorescent proteins exhibiting yellow and orange emission isolated from reef corals. The authors describe six new fluorescent proteins homologous to GFP that share the same beta-barrel structure and demonstrate expression of these variants in mammalian cells and Xenopus embryos.

Bevis, B. J., and Glick, B. S.

Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed).  Nature Biotechnology 20: 83-87 (2002).  Addressing problems with wild-type DsRed fluorescent protein, the authors report mutagenesis efforts to create variants with faster maturation and suppression of green emission. In addition, new mutations were introduced to reduce the net charge near the N-terminus of the protein.

Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., and Tsien, R. Y.

A monomeric red fluorescent protein.  Proceedings of the National Academy of Sciences (USA) 99: 7877-7882 (2002).  A heroic effort to break the tetrameric DsRed fluorescent protein into a monomeric unit that can be used as a stand-alone fusion tag in fluorescence microscopy. The authors report a dimer containing 17 mutations and a monomer named mRFP1 that features 33 mutations compared to wild-type DsRed.

Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., and Tsien, R. Y.

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.  Nature Biotechnology 22: 1567-1572 (2004).  The first report of what has now been termed the "mFruit" series of fluorescent proteins derived from mRFP1. Nathan Shaner and associates describe mCherry, mOrange, tdTomato, mStrawberry and several additional variants.

Merzlyak, E. M., Goedhart, J., Shcherbo, S., Bulina, M. E., Shcheglov, A. S., Fradkov, A. F., Gaintzeva, A., Lukyanov, K. A., Lukyanov, S., Gadella, T. W. J., and Chudakov, D. M.

Bright monomeric red fluorescent protein with an extended fluorescence lifetime.  Nature methods 4: 555-557 (2007).  The authors describe TagRFP, a monomeric orange-red fluorescent protein exhibiting high brightness, complete chromophore maturation, and high pH stability.

Wang, L., Jackson, W. C., Steinbach, P. A., and Tsien, R. Y.

Evolution of new nonantibody proteins via iterative somatic hypermutation.  Proceedings of the National Academy of Sciences (USA) 101: 16745-16749 (2004).  Development of a novel technique known as somatic hypermutation to create monomeric red fluorescent proteins with increased photostability and far-red emission, thus surpassing the best efforts of structure-based design.

Shaner, N. C., Lin, M. Z., McKeown, M. R., Steinbach, P. A., Hazelwood, K. L., Davidson, M. W., and Tsien R. Y.

Improving the photostability of bright monomeric orange and red fluorescent proteins.  Nature Methods 5: 545-551 (2008).  Additional mutagenesis efforts on mOrange and TagRFP to produce variants having improved photostability. The authors introduce a new screen targeting photostability and succeed in producing several of the most photostable fluorescent proteins yet reported.

Karasawa, S., Araki, T., Nagai, T., Mizuno, H., and Miyawaki, A.

Cyan-emitting and orange-emitting fluorescent proteins as pairs for fluorescence resonance energy transfer.  Biochemical Journal 381: 307-312 (2004).  A description of dimeric MiCy, a cyan fluorescent protein, and monomeric Kusabira Orange, a yellow-orange fluorescent protein. The authors discuss mutagenesis efforts to develop these variants and demonstrate their utility in FRET experiments.

Shcherbo, D., Merzlyak, E., Chepurnykh, T., Fradkov, A., Ermakova, G., Solovieva, E., Lukyanov, K., Bogdanova, E., Zaraisky, A., Lukyanov, S., and Chudakov, D.

Bright far-red fluorescent protein for whole-body imaging.  Nature Methods 4: 741-746 (2007).  Report on mutagenesis of TagRFP to generate the far-red fluorescent protein dimeric Katushka as well as a monomeric version named mKate. Both proteins are bright and photostable and potentially useful for tagging and imaging in the far-red portion of the spectrum.

Kredel, S., Oswald, F., Nienhaus, K., Deuschle, K., Rocker, C., Wolff, M., Heilker, R., Nienhaus, G. U., and Wiedenmann, J.

mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures.  PLoS ONE 4: e4391 (2009).  The authors describe mutagenesis efforts to disrupt the tetrameric structure of eqFP611 to generate a new monomeric variant termed mRuby. The new fluorescent protein has an emission profile similar to mCherry and a large Stokes shift of 47 nanometers.