Kaede for Detection of Protein Oligomerization Heike Wolf, B. George Barisas, Karl-Josef Dietz, Thorsten Seidel  Molecular Plant  Volume 6, Issue 5, Pages 1453-1462 (September 2013) DOI: 10.1093/mp/sst039 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Plasmids for Kaede Expression in Plant Cells. (A) The plasmid 35S-Kaede-NosT was designed for transient expression in plant cells. Expression is driven by the CaMV35S promoter. A conventional multiple cloning site is located between promoter and Kaede. The plasmid also carries the Nos-Terminator and an ampicillin resistance gene (ampr). (B) 101-Kaede-HA allows for Gateway® cloning and is designed for stable transformation of plants. The Gateway site is located between the CaMV35S promoter and the coding sequence of Kaede and contains a chloramphenicol resistance gene (CmR) and the ccdB killer gene. Kaede is fused with a HA-tag. The vector is derived from pEarleyGate101. Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Spectral Overlap between Green and Red Form as Prerequisite of FRET. Excitation spectra for recombinant Kaede (blue, green form; orange, red form) and emission spectra (green, green form; red, red form) were recorded for both Kaede forms. The spectral overlap of emission of the green form and excitation of the red form is given in black as the product of the excitation of the red form and emission of the green form. For determination of R0, the absorbance of the red form was used. Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Stability of Kaede and Dynamics of Photoconversion. Kaede was illuminated with 405nm in the fluorometer and emission spectra were repeatedly recorded at defined time points. Kaede was excited at 430nm. The red emission peaking at 576nm increases at the expense of the green emission at 515nm. As it is visible in the spectra, complete conversion was not achieved. Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Influence of Laser Intensity on Photo Conversion. Images of plant cells were obtained by sequential scans with a confocal microscope and emission was quantified along a series of 30 frames. Kaede was photoconverted with a 405-nm diode laser and an intensity of 0 µW, 5.5 µW, 12 µW, 20 µW, 73 µW, and 105 µW, respectively. The emission ratio of the green and red form of Kaede was calculated and the ratio of the first frame was set to 100. Each measurement was repeated three times and the mean average is shown. Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Distribution of RA and Ratio of the Green and Red Form of Kaede along Series of 30 Frames. The frames have been obtained by sequential scanning of green form of Kaede, red form of Kaede, and photoconversion with a confocal laser scanning microscope. (A) The sensitized acceptor emission-related ratio RA has been plotted against the number of recorded frames. The RA curve of Kaede is given in black, the RA curve for 2-cys prx in gray. (B, C) The ratios of green to red form of Kaede (B) and 2-cys prx–Kaede (C) decreased with increasing number of frames and cycles of photoconversion. Representative curves are shown. Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Comparison of RA Values for Various Kaede Constructs. Protoplasts expressed the constructs Kaede, Kaede–Kaede, Gos12-Kaede, and 2-cys prx–Kaede and the corresponding RA values were obtained, respectively. Each experiment was performed twice. Data were pooled except of the 2-cys prx–Kaede measurements. Furthermore, Kaede as well as 2-cys prx were analyzed in the presence of 1 mM reduced DTT (‘reduced’) and subsequent incubation with 1 mM reduced DTT followed by incubation with 10 mM oxidized DTT (‘re-oxidized’). Mean ± SD is given. Asterisks mark highly significant differences of connected samples (Student’s t-test). Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Subcellular Localization of 2-cys prx–Kaede and Kaede Itself. Images of cells expressing either Kaede or 2-cys prx–Kaede were obtained by confocal laser scanning microscopy. Kaede is shown in green, chlorophyll autofluorescence in red. Scale bars correspond to 20 µm. (A) Kaede itself is located in the cytosol. (B) If fused to 2-cys prx, Kaede was localized in the chloroplast and showed a punctuate pattern. (C) Based on images obtained by confocal laser scanning microscopy, correlation analysis of green and red emission of 2-cys prx–Kaede was applied to efficiently separate Kaede from autofluorescence. The emissions of both forms were plotted pairwise against each other. Autofluorescence-derived emission was marked by ellipses, whereas Kaede-derived emission is characterized by significant and correlated emission in both channels. Molecular Plant 2013 6, 1453-1462DOI: (10.1093/mp/sst039) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions