12. M.Z. Lin, M.R. McKeown, H.-L. Ng, T.A. Aguilera, N.C. Shaner, R.E. Campbell, S.R. Adams, L.A. Gross, W. Ma, T. Alber, R.Y. Tsien*, “Autofluorescent Proteins with Excitation in the Optical Window for Intravital Imaging in Mammals”, Chemistry & Biology, 2009, 16: 1169-1179.

Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the “optical window” above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.

11. N.C. Shaner, R.E. Campbell, P.A. Steinbach, B.N.G. Giepmans, A.E. Palmer, and R.Y. Tsien*, “Improved monomeric red, orange, and yellow fluorescent proteins derived from Discosoma red fluorescent protein”, Nat. Biotechnol., 2004, 22: 1567-1572.

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology1, 2. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function3. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive4, 5. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein “DsRed” by directed evolution first to increase the speed of maturation6, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions7. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

10. J. Zhang, R.E. Campbell, A.Y. Ting and R.Y. Tsien*, “Creating New Fluorescent Probes for Cell Biology”, Nat. Rev. Mol. Cell Biol., 2002, 3: 906-918.

Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein–protein interactions.

9. R.E. Campbell, O. Tour, A.E. Palmer, P.A. Steinbach, G.S. Baird, D.A. Zacharias and R.Y. Tsien*, “A Monomeric Red Fluorescent Protein”, Proc. Natl. Acad. Sci. USA., 2002, 99: 7877-7882.

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

8. S.R. Adams, R.E. Campbell, L.A. Gross, B.R. Martin, G.K. Walkup, Y. Yao, J. Llopis and R.Y. Tsien*, “New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications”, J. Am. Chem. Soc., 2002, 124: 6063-6076.

We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269−272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565−578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants (10-11 M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed α-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.

7. O. Griesbeck, G.S. Baird, R.E. Campbell, D.A. Zacharias and R.Y. Tsien*, “Reducing the Environmental Sensitivity of Yellow Fluorescent Protein: Mechanism and Applications”, J. Biol. Chem., 2001, 276: 29188-29194.

Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 °C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK a (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 °C and in organelles. The halide resistance is explained by a 2.2-Å x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca2+] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.