Figure 5. Lifetime measurements of SthK_Cterm-S361Acd-V416C with [Fe(phenM)₃]²⁺ and [Ru(bpy)₂phenM]²⁺.

Chemical structures of acceptors and legends for all plots shown on top. (A–B) Representative Weber plots of phase delay and modulation ratio for SthK_Cterm-S361Acd-V416C labeled with [Fe(phenM)₃]²⁺ (A) and [Ru(bpy)₂phenM]²⁺(B). Fits of the data using the single Gaussian model are shown with phase delay as solid curves and modulation ratio as dashed curves. (C–D) Spaghetti plots showing distance distributions from the model fits with apo (thin black curves, n=4), with 1.23 mM cAMP (thin red curves, n=4) and 1.23 mM cGMP (thin green curves, n=4) for [Fe(phenM)₃]²⁺ (C) and [Ru(bpy)₂phenM]²⁺(D). Distributions predicted by chiLife are overlayed in dashed curves. (E–F) Summary of Gaussian fit standard deviations, $\sigma$, versus average distances, $\overline{r}$, for [Fe(phenM)₃]²⁺ (E) and [Ru(bpy)₂phenM]²⁺ (F), with average values as cross marks.

Figure 5—figure supplement 1. Representative Weber plot of measured lifetimes of SthK_Cterm-S361Acd donor-only (grey), then in the presence of [Fe(phenM)₃]²⁺ (black), and after the addition of 1.23 mM cAMP (red), showing no change in lifetimes in the absence of a cysteine residue.

Figure 5—figure supplement 2. Parameters used in the lifetimes fitting model for both the single and sum of two Gaussian approaches, shown in blue.

(A) Graph of donor-only fluorescence-lifetime decay with two exponential components with time constants (${\tau }_{D1}$, ${\tau }_{D2})$ (B) FRET efficiency (E) plot as a function of distance (r) between donor and acceptor and the R₀ values for 50% FRET transfer. (C) Probability distribution plot of donor and acceptor distances P(r) showing the sum of two Gaussian distributions, each with their own average distance (${\displaystyle \overline{r_1}}$ and ${\displaystyle \overline{r_2}}$), standard deviations (${\sigma }_1$ and ${\sigma }_2$) and relative amplitude of the second component (A₂). The donor-only fraction ($f_D$) is modeled as a narrow Gaussian with mean distance of 150 Å and standard deviation of 0.1 Å, beyond a detectable amount of FRET. (D) The in-phase ($D_{\omega }$) and out-of-phase ($N_{\omega }$) components are plotted for the measured, corrected, and background fluorescence signal as a function of the excitation modulation frequency (ω), where $f_B$ is the fraction of the fluorescence intensity due to the background. (E) Plot of the phase delay (${\phi }_{\omega }$) and modulation ratio ($m_{\omega }$) of the measured and corrected fluorescence response as a function of the modulation frequency (ω) where t₀ is the time shift of the instrument response function.