Figure 3
Structural characterization of the labeled dimer p53 mutants in solution and in FL-p53 droplets using FRET. (a) Schematic diagram of the primary and tertiary structures of the dimer mutant L344A. The cysteines were introduced either at position 292 or at position 394 for labeling with Alexa488 and Alexa594. (b) Fluorescence spectral changes observed after mixing the Alexa488-Alexa488 dimer and the Alexa594-Alexa594 dimer, both labeled at 292 C at pH 7.9 in the presence of 50 mM KCl. The different times describe the periods after mixing. (c) Time course of the spectrum-based FRET efficiency, ES, after mixing the Alexa488-Alexa488 dimer and the Alexa594-Alexa594 dimer labeled at 292 C. The data shown in panel (b) were used for ES calculation. (d) DIC and fluorescence microscopic images of the samples containing 0.12 μM of the Alexa488-Alexa488 dimer labeled at 292 C and 12 μM of FL-p53 (upper panels) at pH 7.0 in the presence of 45 mM NaCl and 150 mg/mL dextran. The corresponding DIC and fluorescence images prepared by adding the Alexa594-Alexa594 dimer labeled at 292 C in the same condition are shown in the lower panels. The fluorescence images were obtained through excitation at 470–490 nm for Alexa488 and at 520–550 nm for Alexa594. (e) DIC and fluorescence microscopic images of the sample after adding the Alexa488-Alexa594 dimer labeled at 292 C to the preformed FL-p53 droplets (upper panels) at pH 7.0 in the presence of 45 mM NaCl and 150 mg/mL dextran. The corresponding DIC and fluorescence images prepared by adding the Alexa488-Alexa594 dimer labeled at 394 C in the same conditions are shown in the lower panels. The fluorescence image was obtained by excitation at 470–490 nm. (f) Comparison between the apparent FRET efficiencies based on the microscopic data, EM, of the Alexa488-Alexa594 dimer labeled at 292 C in solution (left) and in the droplets (right). (g) Comparison between the EM for the Alexa488-Alexa594 dimer labeled at 394 C in solution (left) and in the droplets (right). Error bars are the standard deviation. Scale bars in panels (d,e) represent 10 μm.
