Figure 5. Acd in MBP-295Acd is nonexponential and introduction of the Y307F mutation increases its lifetime and makes it single exponential.

(A, C) Frequency-domain measurements of fluorescence lifetime of (A) MBP-295Acd and (C) MBP-295Acd-Y307F. Black, phase delay in degrees; red, modulation ratio. Curves are fits with Equations 5 and 6 corrected by Equations 13 and 14. Fit parameters for (A) are ${\tau }_{D_1}$ = 11.6 ns for a single exponential (solid curve) and ${\tau }_{D_1}$ = 13.9 ns, ${\tau }_{D_2}$ = 1.9 ns, and α₁ = 0.70 Å (dashed curve) and for (C) are ${\tau }_{D_1}$ = 15.6 ns. (B) Cartoon representation of the proximity between Acd at position 295 and tyrosine at position 307 in the apo state. (D, E) Collected data from fits for MBP-295Acd (WT) and MBP-295Acd-Y307F, as indicated. Independent samples are shown as red circles, and the means are shown as black lines.

Figure 5—figure supplement 1. Steady-state fluorescence to measure transition metal ion fluorescence resonance energy transfer (tmFRET) between MBP-295Acd-Y307F-C and Cu²⁺-TETAC.

(A) Quenching by Cu²⁺-TETAC and recovery with TCEP for MBP-295Acd-Y307F-C, with F_Cys and F_{no Cys} as defined in Equation 2. Data are shown as mean ± SEM for n = 5. 10 µM Cu²⁺-TETAC and 2.5 mM TCEP were added at the times indicated by the bars. Open symbols represent data collected in the absence of maltose, and filled symbols represent data collected in the presence of 10 mM maltose. (B) Collected data showing each independent sample (circles) and the mean (red line) for F_Cys and F_{no Cys}. The absence and presence of 10 mM maltose are as indicated by the – and + shown beneath each data set.