Structure of SthK constructs and modular gating scheme.

(A) Cartoon structures for SthKCterm and SthKFull with the domains labeled on the SthKFull structure. (B) Modular gating scheme for SthK showing modules corresponding to domains indicated in the structures above. Within each domain, resting (R) and active (A) or closed (C) and open (O) or unbound (U) and bound (B) conformations are shown connected by double arrows, representing the transition between states. Horizontal arrows between modules indicate the coupling between domains.

FRET with the donor-acceptor pair within the same subunit.

(A) Cartoon of SthKCterm:WT/S361Acd-V416C with a single donor-acceptor labeled subunit (top; acceptor shown as magenta dot) and the structures of donor fluorophore, Acd, and acceptor metal complex [Ru(bpy)2phenM]2+ (bottom). (B) Representative TCSPC data from SthKCterm:WT/S361Acd treated with [Ru(bpy)2phenM]2+ before (grey) and after (orange) the addition of 320 μM cAMP. TCSPC data from SthKCterm:WT/S361Acd-V416C treated with [Ru(bpy)2phenM]2+ in the absence of cAMP (black) and in the presence of 1 μM (blue) or 320 μM (red) cAMP. Smooth black curves show the fits of the FRET model, and weighted residuals are shown below using the same colors as used for the data. Representative instrument response function (IRF) shown in purple. (C) Summary of distance parameter values, and σ (n = 4), from fits to our TCSPC data (filled symbols) and from previously published frequency domain data (open symbols), with resting and active state as indicated in the legend (12). (D) fA2 values determine from fits to TCSPC data measured with 0, 1 μM and 320 μM cAMP (filled symbols) compared to previously published fA2 values determine from frequency domain data (open symbols) (12). (E) Spaghetti plot of distance distributions from individual experiments for 0 (black), 1 μM (blue) and 230 μM (red) cAMP, with distributions predicted by chiLife shown as dashed curves. Resting and active are shown in black and red, respectively. (F) Summary of fA2 values over range of cAMP concentrations. The dose-response relation was fit with a quadratic equation (red curve) with KD = 0.22 μM and [P]total = 1.2 μM.

FRET with the donor-acceptor pair on different subunits.

(A) Cartoon structure of SthKCterm:V416C/ S361Acd protein with a single donor-labeled subunit (SthKCterm:S361Acd) and 3 acceptor-labeled subunits (SthKCterm:V416C). (B) Representative TCSPC data from the donor-only condition (grey), and after [Ru(bpy)2phenM]2+ labeling either in the absence (black) or presence (red) of 320 μM cAMP, fit with the FRET model (smooth black lines). The IRF and weighted residuals are depicted as in Figure 2. (C) Summary of distance parameter values, and σ (n = 4), from fits of our TCSPC data with the FRET model. Resting and active state are shown as black and red, respectively. (D) Distributions of donor-acceptor distances predicted with chiLife for intrasubunit distances (darker dashed curves) and intersubunit distances (lighter dashed curves), with black and red as described in (C). The distance distributions from fits to our TCSPC data for resting and active states are overlayed as solid black and red Gaussians.

FRET with both intrasubunit and intersubunit donor-acceptor pairs in SthKCterm.

(A) Cartoon structure of homotetrameric SthKCterm:S361Acd-V416C protein. (B) Representative TCSPC data from the donor-only condition (in grey) and after [Ru(Bpy)2phenM]2+ labeling in the absence of cAMP (black) and in the presence of either 1 μM (blue) or 320 μM (red) cAMP, fit with the FRET model (smooth black lines). The IRF and weighted residuals are depicted as in Figure 2. (C) Summary of distance parameter values, and σ (n = 4), from fits of our TCSPC data with the FRET model. Resting and active state are shown as black and red, respectively. (D) Values of fA2 for 0, 1 μM or 320 μM cAMP, as indicated. (E) Spaghetti plot of distance distributions from individual experiments for 0 (black), 1 μM (blue) and 230 μM (red) cAMP, with distributions predicted by chiLife shown as dashed curves. Resting and active are indicated by black and red, respectively.

Expression and purification of SthKFull incorporating Acd.

(A) SDS/PAGE with in-gel fluorescence (left) or Coomassie blue (right) for SthKCterm:S361Acd and SthKCterm:S361Acd-V416C, as indicated (theoretical mass = 53.0 kDa). (B) Chromatogram from size exclusion chromatography monitoring fluorescence at 425 nm (Acd) for SthKCterm:S361Acd-V416C.

FRET with both intrasubunit and intersubunit donor-acceptor pairs in SthKFull.

(A) Cartoon structure of SthKFull:S361Acd-V416C construct. (B) Representative TCSPC data from the donor-only condition (no cAMP – gray, 320 µM cAMP – orange) and SthKFull:S361Acd-V416C after [Ru(bpy)2phenM]2+ labeling in the absence of cAMP (black) and in the presence of either 1 μM (blue) or 320 μM (red) cAMP, fit with the FRET model (smooth black lines). The IRF and weighted residuals are depicted as in Figure 2. (C) Summary of distance parameter values, and σ (n = 4), from fits of our TCSPC data with the FRET model. Resting and active state are shown as black and red, respectively. (D) Values of fA2 for 0, 1 μM or 320 μM cAMP, as indicated. (E) Spaghetti plot of distance distributions from individual experiments for 0 (black), 1 μM (blue) and 230 μM (red) cAMP, with distributions predicted by chiLife shown as dashed curves. Resting and active are indicated by black and red, respectively. (F) Summary of fA2 over range of cAMP concentrations. The dose-response relation was fit with a quadratic equation (red curve) with KD = 0.53 μM and [P]total = 0.025μM. (G-H) Values of fA2 from globally fitting SthKFull and SthKCterm constructs.

Energetics of the resting to active conformational change in the CNBD.

A hypothetical reaction coordinate shows the relative energy difference between resting and active states (ΔG) and difference in free energy change (ΔΔG) without ligand (black) and with cAMP bound (red), for SthKCterm (left) and SthKFull (right). Calculated ΔG and ΔΔGs values are shown in for each construct below the diagram.

Parameters used in the FRET model, shown in blue, for TCSPC data for the sum of two Gaussians distribution.

(A) Graph of donor-only fluorescence-lifetime decay with two exponential components with time constants (τD1, τD2). The amplitude fraction of the donor-only lifetime in an experiment is determined as fD. (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 ( and), standard deviations (σ1 and σ2) and relative amplitude of the second component (fA2). (D) Example TCSPC data is shown with experimental measured photon count data (Decay), the instrument response function (IRF), and the buffer only decay (DecayB), with its corresponding scaling factor fB relative to the Decay trace. Also indicated is a time-independent background of photon counts for the Decay trace, bkgrdee, as well as two parameters associated with the IRF, its background, bkgrirf, and the shift between instrument response function and measured decay, shiftirf. Parameter AO is the amplitude of the FRET model estimate of the fluorescence lifetime of the donor (in photon counts).

A) Identifiability of parameters in the sum of two Gaussian distributions model for representative TCSPC data. (A) Dependence of the reduced χ2 values on the fraction of donor-only component (fD). (B-C) Dependence of the reduced χ2 values on the Gaussian average distances (B) and standard deviations (σ) (C) for the resting (black) and active states (red). (D) Dependence of the reduced χ2 values on the parameter fA2 for the conditions of apo, 0.5 μM, 1 μM and 320 μM cAMP.

(A-F) Parameter correlations for representative TCSPC data of χ2 values in 3 dimensions. Plots of χ2 values vs. σ1 and fD (A), vs. σ2 and fD (B), vs. fA2 in 0 μM cAMP and fD (C), vs. fA2 in 320 μM cAMP and fD (D), vs σ1 and (E) and vs. σ2 and (F).