Characterization of the dual-FRET biosensor, CYR83.

<p>(<b>A</b>) A schematic structure of a FRET-based biosensor, CYR83. CYR83 consists of three fluorescent proteins, seCFP (CFP), Venus and mRFP1 (RFP); the linker portion contains two distinct caspase cleavage sequences, IETD and DEVD. (<b>B</b>) Induction of a conformational change of CYR83 by active caspases. The emission spectra of CYR83 were measured by exciting at 440 nm (left panels) or 500 nm (right panels) with a spectrophotometer before or at indicated time of incubation with recombinant active CASP8 (upper panels) or active CASP3 (lower panels). Arrows indicate the major peaks of fluorescent proteins (seCFP, Venus and mRFP1), and the direction reveals the dynamics of the fluorescence intensity. The numbers in the inset box indicate the emission ratio on 528 nm/476 nm and 608 nm/476 nm or 608 nm/528 nm before and after 120 min of incubation with active caspases. (<b>C</b>, <b>D</b>) <i>In vitro</i> cleavage assay of CYR83 by active caspases. Fluorescence data shown in panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s001" target="_blank">Figure S1A</a>–F were merged and represented as (C) and (D). Lower-case characters indicate full length CYR83 (a) and the cleaved peptide fragments (b-f) during incubation with active caspases. Numbers indicate incubation time. Abbreviations; M, FITC-conjugated molecular weight markers; C, seCFP; V, Venus; R, mRFP1. (<b>E</b>) Profiles of the products cleaved by active caspases. Fluorescent bands corresponding to the processed seCFP, Venus, mRFP1, seCFP-Venus and Venus-mRFP1 and the intact CYR83 were extracted from the fluorescence images shown in (C) and (D).