Autofluorescent Proteins with Excitation in the Optical ... - Tsien

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Animal Imaging Transcription units containing mKate, dNeptune, or IFP1.1 followed by the poliovirus IRES and a GFP coding sequence were constructed in pENTR1 by standard methods, and then recombined into pAd-CMV-DEST by the Gateway system (Invitrogen). Linearized plasmid was used to transfect HEK293A cells for adenovirus production, followed by amplification and purification on FastTrap columns (Millipore) and elution into HBSS + 10% glycerol. Serial dilutions onto HEK293A cells and GFP visualization were performed to assess titers. Injections, imaging, and dissection were carried out in duplicate. Mice were anesthesized with ketamine/xylazine, and then injected via the tail vein with 50 mlof53 10 10 infectious units per milliliter of purified adenoviruses. Five days later, mice were shaved on their ventral side and anesthesized with ketamine/xylazine. Images were acquired on a Maestro 1 animal fluorescence imaging system (CRI) with indicated excitation filters and a tunable liquid crystal emission filter of 40 nm bandwidth centered on the indicated wavelengths. For quantification, off-animal dark counts were first subtracted, and then mid-sagittal line intensities were obtained in ImageJ with the Plot Profile function. Similar results were obtained in the duplicate samples for each condition. For the charts in Figure 3, line intensities from duplicate conditions were averaged and background was defined as the mean mid-sagittal intensity of uninfected liver, treatment-matched in terms of pre- or post-dissection condition. Defining background as the mid-thoracic region of infected animals gave similar results. For confocal imaging, three randomly oriented freshly prepared liver slices were placed on a coverslip and imaged with a 403 water objective on a Zeiss LSM 5Live microscope. For each slice, fluorescence intensities were measured from maximum intensity projections of a ten section stack of total thickness of 40 mm. All animal procedures were performed according to protocols approved by the University of California, San Diego, institutional animal care and use committee. For spectral unmixing, the predicted Neptune distribution in the infrared image is removed based on the Neptune distribution in the far-red channel and the relative efficiencies in the two channels in detecting Neptune, as calculated from the Neptune spectra. Crystallization and Refinement For crystallization, the His tag on the protein was removed by TEV protease digestion. Crystals were grown by the sitting drop vapor diffusion method. One microliter of protein at 10 mg/ml was mixed with 1 ml of 0.1 M HEPES (pH 7.0), 0.2 M CaCl2, and 20% PEG 6000 at 18 C. Crystals were cryoprotected with a mixture of 66.5% Paratone-N, 28.5% paraffin oil, and 5% glycerol (Sugahara and Kunishima, 2006) prior to immersion in liquid nitrogen. Data were collected at Beamline 8.3.1 at the Advanced Light Source, Lawrence Berkeley National Laboratory and processed with ELVES (Holton and Alber, 2004) and MOSFLM/SCALA (Evans, 2006; Leslie, 2006). The crystal structure was solved by molecular replacement using PHASER (McCoy et al., 2007) with mKate at pH 4.2 without the chromophore (PDB 3BXA) as the search model. The structure was refined with TLS parameters using PHENIX (Zwart et al., 2008), alternating with manual revision of the model using COOT (Emsley and Cowtan, 2004). No planarity restraints were applied to the chromophore during refinement. Crystallographic data and refinement statistics are presented in Table 3. Structure validation was performed with WHAT_CHECK (Hooft et al., 1996). Neptune and mKate (pH 7.0) (PDB file 3BXB) were aligned and root mean square deviations were calculated using the align command in PyMol (http://www.pymol.org). ACCESSION NUMBERS Coordinates have been deposited in the PDB with accession codes 3IP2. Sequences have been deposited in the EMBL Nucleotide Sequence Database with accession codes FN565568 (Neptune) and FN565569 (mNeptune), and in GenBank with accession codes GU189532 (Neptune) and GU189533 (mNeptune). SUPPLEMENTAL DATA Supplemental Data include Supplemental Experimental Procedures, six figures, and four tables and can be found with this article online at http:// www.cell.com/chemistry-biology/supplemental/S1074-5521(09)00360-3. ACKNOWLEDGMENTS We thank Paul Steinbach for assisting with protein photobleaching and related analysis. We acknowledge the following sources of support: the Jane Coffin Childs Foundation and a Burroughs Wellcome Career Award for Medical Scientists (M.Z.L.), the University of California, San Diego, Chancellor’s Award for Undergraduate Research (M.R.M.), a Howard Hughes Medical Institute (HHMI) predoctoral fellowship (N.C.S.), a Canadian Institutes of Health Research postdoctoral fellowship (R.E.C.), the National Institutes of Health (NIH) Medical Scientist Training Program and U.S. Army Breast Cancer Research Program (BCRP) grant W81XWH-05-01-0183 (T.A.A.), NIH grant R01 GM048958 (H-L.N., W.M., and T.A.), HHMI, NIH grants R01 GM086197 and R37 NS027177, and BCRP grant W81XWH-05-01-0183 (R.Y.T.). Received: June 29, 2009 Revised: October 5, 2009 Accepted: October 12, 2009 Published: November 24, 2009 REFERENCES 1178 Chemistry & Biology 16, 1169–1179, November 25, 2009 ª2009 Elsevier Ltd All rights reserved Chemistry & Biology Fluorescent Proteins with Far-Red Excitation Abbyad, P., Childs, W., Shi, X., and Boxer, S.G. (2007). Dynamic Stokes shift in green fluorescent protein variants. Proc. Natl. Acad. Sci. USA 104, 20189–20194. Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989. Chalfie, M., and Kain, S.R. (2006). Green Fluorescent Protein: Properties, Applications, and Protocols (Hoboken, NJ: Wiley-Interscience). Colin, M., Moritz, S., Schneider, H., Capeau, J., Coutelle, C., and Brahimi- Horn, M.C. (2000). Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo. Gene Ther. 7, 1333–1336. Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. Evans, P. (2006). Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82. Gross, L.A., Baird, G.S., Hoffman, R.C., Baldridge, K.K., and Tsien, R.Y. (2000). The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11990–11995. Henderson, J.N., Ai, H.W., Campbell, R.E., and Remington, S.J. (2007). Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc. Natl. Acad. Sci. USA 104, 6672–6677. Holton, J., and Alber, T. (2004). Automated protein crystal structure determination using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542. Hooft, R.W., Vriend, G., Sander, C., and Abola, E.E. (1996). Errors in protein structures. Nature 381, 272. Lakowicz, J.R. (2006). Principles of Fluorescence Spectroscopy (New York: Springer). Leslie, A.G. (2006). The integration of macromolecular diffraction data. Acta Crystallogr. D Biol. Crystallogr. 62, 48–57. Li, H., Robertson, A.D., and Jensen, J.H. (2005). Very fast empirical prediction and rationalization of protein pKa values. Proteins 61, 704–721. McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674. Ntziachristos, V. (2006). Fluorescence molecular imaging. Annu. Rev. Biomed. Eng. 8, 1–33. Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., and Remington, S.J. (1996). Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395. Petersen, J., Wilmann, P.G., Beddoe, T., Oakley, A.J., Devenish, R.J., Prescott, M., and Rossjohn, J. (2003). The 2.0-A crystal structure of eqFP611,
Chemistry & Biology Fluorescent Proteins with Far-Red Excitation a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J. Biol. Chem. 278, 44626–44631. Pletnev, S., Shcherbo, D., Chudakov, D.M., Pletneva, N., Merzlyak, E.M., Wlodawer, A., Dauter, Z., and Pletnev, V. (2008). A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore. J. Biol. Chem. 283, 28980–28987. Remington, S.J. (2006). Fluorescent proteins: maturation, photochemistry and photophysics. Curr. Opin. Struct. Biol. 16, 714–721. Remington, S.J., Wachter, R.M., Yarbrough, D.K., Branchaud, B., Anderson, D.C., Kallio, K., and Lukyanov, K.A. (2005). zFP538, a yellow-fluorescent protein from Zoanthus, contains a novel three-ring chromophore. Biochemistry 44, 202–212. Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572. Shaner, N.C., Steinbach, P.A., and Tsien, R.Y. (2005). A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909. Shaner, N.C., Lin, M.Z., McKeown, M.R., Steinbach, P.A., Hazelwood, K.L., Davidson, M.W., and Tsien, R.Y. (2008). Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551. Shcherbo, D., Merzlyak, E.M., Chepurnykh, T.V., Fradkov, A.F., Ermakova, G.V., Solovieva, E.A., Lukyanov, K.A., Bogdanova, E.A., Zaraisky, A.G., Lukyanov, S., and Chudakov, D.M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4, 741–746. Shcherbo, D., Murphy, C.S., Ermakova, G.V., Solovieva, E.A., Chepurnykh, T.V., Shcheglov, A.S., Verkhusha, V.V., Pletnev, V.Z., Hazelwood, K.L., Roche, P.M., et al. (2009). Far-red fluorescent tags for protein imaging in living tissues. Biochem J. 418, 567–574. Shu, X., Shaner, N.C., Yarbrough, C.A., Tsien, R.Y., and Remington, S.J. (2006). Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647. Shu, X., Royant, A., Lin, M.Z., Aguilera, T.A., Lev-Ram, V., Steinbach, P.A., and Tsien, R.Y. (2009a). Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807. Shu, X., Wang, L., Colip, L., Kallio, K., and Remington, S.J. (2009b). Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein. Protein Sci. 18, 460–466. Sniegowski, J.A., Lappe, J.W., Patel, H.N., Huffman, H.A., and Wachter, R.M. (2005). Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein. J. Biol. Chem. 280, 26248–26255. Stamatas, G.N., Southall, M., and Kollias, N. (2006). In vivo monitoring of cutaneous edema using spectral imaging in the visible and near infrared. J. Invest. Dermatol. 126, 1753–1760. Sugahara, M., and Kunishima, N. (2006). Novel versatile cryoprotectants for heavy-atom derivatization of protein crystals. Acta Crystallogr. D Biol. Crystallogr. 62, 520–526. Taguchi, N., Mochizuki, Y., Nakano, T., Amari, S., Fukuzawa, K., Ishikawa, T., Sakurai, M., and Tanaka, S. (2009). Fragment molecular orbital calculations on red fluorescent proteins (DsRed and mFruits). J. Phys. Chem. B 113, 1153–1161. Tromberg, B.J., Shah, N., Lanning, R., Cerussi, A., Espinoza, J., Pham, T., Svaasand, L., and Butler, J. (2000). Non-invasive in vivo characterization of breast tumors using photon migration spectroscopy. Neoplasia 2, 26–40. Wang, L., Jackson, W.C., Steinbach, P.A., and Tsien, R.Y. (2004). Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 16745–16749. Zwart, P.H., Afonine, P.V., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., McKee, E., Moriarty, N.W., Read, R.J., Sacchettini, J.C., et al. (2008). Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435. Chemistry & Biology 16, 1169–1179, November 25, 2009 ª2009 Elsevier Ltd All rights reserved 1179
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