Near-infrared fluorescent proteins

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1 nature methods Near-infrared fluorescent proteins Dmitry Shcherbo, Irina I Shemiakina, Anastasiya V Ryabova, Kathryn E Luker, Bradley T Schmidt, Ekaterina A Souslova, Tatiana V Gorodnicheva, Lydia Strukova, Konstantin M Shidlovskiy, Olga V Britanova, Andrey G Zaraisky, Konstantin A Lukyanov, Victor B Loschenov, Gary D Luker & Dmitriy M Chudakov Supplementary figures and text: Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Transgenic zebrafish expressing Katushka in muscle cells. Comparison of cytotoxicity of selected fluorescent proteins in E. coli. Comparison of cytotoxicity of Katushka and Katushka-9-5 in HeLa cells. Alignment of the amino acid sequences of selected fluorescent proteins. Comparison of fluorescent properties of selected fluorescent proteins in bacterial streaks. Transient transfection of HeLa cells by eqfp650 and eqfp670. Katushka, eqfp650 and eqfp670 fluorescence and absorbance spectra.

2 Supplementary Figure 1. Transgenic zebrafish expressing Katushka in muscle cells. (a) White light image. (b) Overlay of white light and fluorescence images in a stereomicroscope.

3 mcherry Katushka Katushka 9-5 eqfp650 eqfp670 mneptune E2-Crimson IPTG induction estimated cytotoxicity no induction medium high low low low high low Supplementary Figure 2. Comparison of cytotoxicity of selected fluorescent proteins in E. coli. To compare protein cytotoxicity in prokaryotes, E. coli cells (XL1 Blue strain) were transformed with pqe30 vectors encoding corresponding fluorescent proteins. Equal portions of bacteria were inoculated on LB agar plates with or without 10 µg/ml IPTG to induce high promoter activity. Bacterial clones were photographed after overnight growth at 37 C. It should be noted that expression of fluorescent proteins in XL1 Blue strain from pqe30 vector produces sufficiently high expression levels without IPTG induction, due to the intense promoter leakage. Induction by IPTG further raises expression levels that become cytotoxic for growing E. coli, slowing the rate of bacterial growth and producing smaller colonies. For those proteins that demonstrate relatively high cytotoxicty at the given expression level, colony growth can be completely blocked. Estimated relative cytotoxicity in bacteria is shown in the lower row. Scale bar, 1 cm. 2

4 Supplementary Figure 3. Comparison of cytotoxicity of Katushka and Katushka-9-5 in HeLa cells. (a) Images obtained on days 3, 4 and 5 after transfection with Katushka or Katushka-9-5. Scale bar, 200 μm. (b) The same cells were analyzed by flow cytometry. Graphs show percentage and brightness of cells expressing Katushka or Katushka days after transfection. Columns show percentage of cells expressing Katushka or Katushka and 5 days after transfection, respectively. The same high numbers of living fluorescent cells were detected after five days of culturing by both fluorescence microscopy and flow cytometry. Standard deviations (n = 3 transfection experiments) are shown. 3

5 avgfp MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG-KLPVPWPT DsRed MRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVKLKVTKGGPLPFAWDI E2-Crimson MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKLQVTKGGPLPFAWDI eqfp578 MSELIKENMHMKLYMEGTVNNHHFKCTSEGERKPYEGTQTMKIKVVEGGPLPFAFDI mneptune MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTGRIKVVEGGPLPFAFDI Katushka MGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTMKIKVVEGGPLPFAFDI Katushka-9-5 MGEDSELISENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTMKIKVVEGGPLPFAFDI eqfp650 MGEDSELISENMHMKLYMEGTVNGHHFKCTSEGEGKPYEGTQTAKIKVVEGGPLPFAFDI eqfp670 MGEDSELISENMHTKLYMEGTVNGHHFKCTSEGEGKPYEGTQTCKIKVVEGGPLPFAFDI avgfp LVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT DsRed LSPQFQYGSKVYVKHPADIP--DYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGC E2-Crimson LSPQFFYGSKAYIKHPADIP--DYLKQSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGT eqfp578 LATSFMYGSKTFINHTQGIP--DLFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGC mneptune LATCFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGC Katushka LATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGC Katushka-9-5 LATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGC eqfp650 LATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGC eqfp670 LATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGC avgfp LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQL DsRed FIYKVKFIGVNFPSDGPVMQKKTM-GWEASTERLYPR--DGVLKGEIHKALKLKDGGHYL E2-Crimson LIYHVKFIGVNFPSDGPVMQKKTL-GWEPSTERNYPR--DGVLKGENHMALKLKGGGHYL eqfp578 IIYNVKINGVNFPSNGSVMQKKTL-GWEANTEMLYPA--DGGLRGHSQMALKLVGGGYLH mneptune LIYNVKIRGVNFPSNGPVMQKKTL-GWEASTETLYPA--DGGLEGRCDMALKLVGGGHLI Katushka LIYNVKINGVNFPSNGPVMQKKTL-GWEASTEMLYPA--DSGLRGHSQMALKLVGGGYLH Katushka-9-5 LIYNVKINGVNFPSNGPVMQKKTL-GWEASTEMLYPA--DSGLRGHSQMALKLVGGGYLH eqfp650 LIYNVKINGVNFPSNGPVMQKKTL-GWEASTEMLYPA--DSGLRGHSQMALKLVGGGYLH eqfp670 LIYNVKINGVNFPSNGPVMQKKTL-GWEANTEMLYPA--DSGLRGHNQMALKLVGGGYLH avgfp ADHYQQNTPIGD-GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK DsRed VEFKSIYMAKKP---VQLPGYYYVDSKLDITSH-NEDYTIVEQYERTEGRHHLFL E2-Crimson CEFKSIYMAKKP---VKLPGYHYVDYKLDITSH-NEDYTVVEQYERAEARHHLFQ eqfp578 CSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKE-ADKETYVEQHEMAVAKYCDLPSKLGHR mneptune CNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKE-ADNETYVEQHEVAVARYCDLPSKLGHKLNGMDELYK Katushka CSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS Katushka-9-5 CSLKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS eqfp650 CSLKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS eqfp670 CSLKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS Supplementary Figure 4. Alignment of the amino acid sequences of selected fluorescent proteins. Structurally important regions are highlighted in gray, beta-strands are shown with arrows, and alpha-helices are shown with ribbons. Chromophore-forming residues are underlined. The differences between Katushka and Katushka-9-5 are shown in green. Note that Glu6 was inherited from the wild-type eqfp578. Substitutions that resulted in the formation of eqfp650 and eqfp670 are shown in red (directed changes) or in blue (random mutations). Mutation of Met44 in E2-Crimson and mneptune is shown yellow. Overall alignment numbering follows that of Aequorea victoria GFP (avgfp). 4

6 Supplementary Figure 5. Comparison of fluorescent properties of selected fluorescent proteins in bacterial streaks. Streaks of E. coli transformed with a corresponding fluorescent protein were imaged with a fluorescent stereomicroscope using various filter sets or under 635 nm diodes after overnight growth at 37 C. Note that relatively low brightness of mneptune is characteristic for bacterial cells only. In eukaryotic cells, both mneptune and Neptune demonstrate high brightness 48 h after transfection (see Fig. 1b of the main text). 5

7 eqfp650 eqfp670 Supplementary Figure 6. Transient transfection of HeLa cells by eqfp650 and eqfp670. HeLa cells transiently transfected with eqfp650 or eqfp670 were visualized using widefield Leica AFLX 6000 microscope, 63x objective, after 3 days of incubation. Scale bars, 10 μm. 6

8 A. 1,0 eqfp650 eqfp670 Katushka Fluorescence, a.u. 0,8 0,6 0,4 0, Wavelength, nm B. 1,0 0,8 eqfp650 eqfp670 Katushka Absorbance, a.u. 0,6 0,4 0, Wavelength, nm Supplementary Figure 7. Katushka, eqfp650 and eqfp670 fluorescence and absorbance spectra. (a) Excitation (solid lines) and emission (dashed lines) spectra. (b) Absorbance spectra. 7