Figure 2.

CB FRET sensor design and characterization. (a) CB domain architecture with the SH3, DH, and PH domains in light gray, black, and dark gray, respectively. Amino acid positions Trp24 (W24) and Glu262 (E262) are highlighted. (b) Domain architecture of the ensemble of CB FRET sensors constructed in this study, highlighting the position of the tetra-cysteine motif (tCM) used for labeling with the fluorescein arsenical hairpin binder-ethanedithiol (FlAsH-EDT2) (green spheres) and C-terminal attachment site of CFP (teal). Individual sensors contained a single tCM inserted after residues 1 (F1), 28 (F28), 73 (F73), and 99 (F99), whereas the CFP position (after residue 456) was kept constant. F1_D0, F28_D0, F73_D0, and F99_D0 represent the individual FRET sensors in the absence of FlAsH and F1_DA, F28_DA, F73_DA, and F99_DA after FlAsH labeling. In the single-mutant FRET sensor (F1_smD0) E262 was replaced with Ala (E262A) and in the double-mutant FRET sensor (F1_dmD0), W24 and E262 were replaced with Ala (W24A/E262A). (c) Cartoon representing the CB FRET sensor (F1_D0) in the closed conformation highlighting its labeling with FlAsH-EDT₂ reagent, resulting in F1_DA. FlAsH-EDT₂ (nonfluorescent) turns fluorescent (green) after forming covalent bonds with the cysteine residues present in tCM (green circle). (d) Time-resolved fluorescence intensities of CFP of the CB FRET sensor (F1_D0; teal) and the FlAsH-labeled CB FRET sensor (F1_DA; cyan). The instrument response function (IRF) is shown in gray. F1_D0 and F1_DA were excited (λ_ex) at 440 nm and emission (λ_em) data were collected between 460 and 500 nm. Data were scaled to a maximum of 100 for easier comparison. (e) Species-weighted ⟨τ⟩_x of CFP in F1_DA (1.20 ± 0.04 ns) is reduced compared with F1_D0 (2.16 ± 0.06 ns), corresponding to a FRET efficiency of 44% (Eq. 8). Data from three different batches of experiments are presented as mean values ± SD.