SthK as a model protein for characterizing the allosteric regulation in CNBD channels. (A) Representative macroscopic A208V-cfSthK currents in inside-out patches from bacterial spheroplasts in response to voltage steps shown at the top, in the absence of cyclic nucleotide (top black trace), in saturating 1 mM cAMP (middle red trace), and in saturating 1 mM cGMP (bottom green trace). (B) Dose-response relation of A208V-cfSthK to cAMP at +80 mV (red circles, n=6), fit with the Hill Equation (in red, Kl/2: 0.27 ± 0.01 µM, slope: 2.9 ± 0.2, ± SD). Fractional activation by 1 mM cGMP was 0.002 ± 0.003 (± SD) (green diamond, n=6). (C) State diagram showing four states (either apo or holo, and either resting or active) of the CNBD and the associated ΔG’s for the transitions between states. (D) Theoretical fluorescence lifetime decays of a donor fluorophore in the time-domain showing basis of time-resolved tmFRET. A single exponential donor and mixtures of two single tmFRET distances (short and long) are shown. (E) Theoretical distance distributions showing two states with average distances,r̄, heterogeneity within each conformational state as standard deviation, σ, and heterogeneity between conformational states as fractional area A2 for apo (black) and holo (red) conditions.

Expression, purification, and tetrameric analysis of SthKC-term. (A) In-gel protein fluorescence showing selective Acd incorporation into SthKC-term in the absence and presence of S361Acd TAG site, Acd aminoacyl tRNA synthetase/tRNA (RS/tRNA), and unnatural amino acid Acd. (B) Structures of cysteine modified by acceptor compounds [Cu(TETAC)]2+ (cyan), [Fe(phenM)3]2+ (magenta), and [Ru(bpy)2phenM]2+ (orange) along with their corresponding Förster curves of FRET efficiency as a function of distance from Acd, and their R0 values specified and marked with dashed lines. (C) SthKC-term cartoon as tetramer and monomer showing WT subunits in gray and cysteine-containing Acd-labeled subunits in orange. (D) SEC traces (absorbance at 280 nm in black and 425 nm fluorescence emission for Acd in blue) of isolated WT-Acd-heterotetrameric protein (closed triangle) vs monomeric WT protein (open triangle). (E) Mass photometry histogram data showing primarily tetramers, with single Gaussian fits (blue, 29 kDa ± 7.3 and 104.5 kDa ± 16.8, ± SD).

Steady-state tmFRET data from tetrameric SthKC-term. (A) Left: structure of one subunit of SthKC-term-S361Acd with [Fe(phenM)3]2+ acceptor incorporated at V416C (adapted from PDB: 4D7T) (9). Middle: averaged fluorescence time course upon addition of [Fe(phenM)3]2+ and then cAMP or cGMP (apo, n=8; cAMP n=6; and cGMP, n=2). Right: summary of the FRET efficiencies from individual experiments, with mean values as horizontal lines. (B) Left: Average fluorescence time course for same site upon addition of [Ru(bpy)2phenM]2+ acceptor and then cAMP or cGMP (apo, n=8; cAMP, n=4; and cGMP, n=4). Right: summary of FRET efficiencies, with mean values as horizontal lines. (C) Left: structure of one subunit of SthKC-term-Q364Acd-417C with [Cu(TETAC)]2+ acceptor incorporated at R417C. Middle: averaged fluorescence time course upon addition of [Cu(TETAC)]2+ then cAMP or cGMP (apo, n= 8; cAMP, n=5; and cAMP, n=3). Right: summary of FRET efficiencies, with mean values as horizontal lines. (D) Dose response relations of FRET efficiency change as a function of cAMP concentration normalized for comparison and fit with Hill equations (Kl/2: 0.25 ± 0.01 µM, 0.14 ± 0.01 µM, 0.21 ± 0.02 µM, and h:1.2 ± 0.07, 1, and 1 for [Fe(phenM)3]2+, magenta; [Ru(bpy)2phenM]2+, orange; and [Cu(TETAC)]2+, cyan respectively, ± SD).

Distance distribution predictions with chiLife. (A) Structure of resting state (left, PDB:7RSH) (11), and cAMP-bound active state (right, PDB:4D7T) (9) with rotameric clouds predicted by chiLife for the labels (Acd, blue; [Fe(phenM)3]2+, magenta; and [Cu(TETAC)]2+, cyan). Gray surface indicates location of adjacent WT subunits in tetramer. (B) Distance distributions predicted by chiLife for SthKC-term-S361Acd-V416C-[Fe(phenM)3]2+, SthKC-term-S361Acd-V416C-[Ru(bpy)2phenM]2+ and SthKC-term-Q364Acd-R417C-[Cu(TETAC)]2+ (intra-subunit distances: resting, black curves; active, red curves; and inter-subunit distances: resting, grey curves; active, pink curves). Average steady-state tmFRET distance measurements are overlayed as vertical lines for data from Figure 3 (apo, solid black, and cAMP, solid red) and adjusted assuming a 10% unlabeled protein (dashed black and red vertical lines).

Lifetime measurements of SthKC-term-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 SthKC-term-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, σ, versus average distances, r̄, for [Fe(phenM)3]2+ (E) and [Ru(bpy)2phenM]2+ (F), with average values as cross marks.

Analysis of lifetime data with global fit model allowing sum of two Gaussian distance distributions. (A) Representative phasor plot of measured lifetimes with [Fe(phenM)3]2+ acceptor, where markers on universal circle indicate single-exponential time constants (in nanoseconds). Data shown are donor-only (grey), apo (black), cAMP (red) and cGMP (green). (B) Representative Weber plot showing global fits for [Fe(phenM)3]2+ acceptor data (closed circles) and [Ru(Bpy)2phenM]2+ acceptor data (open diamonds) for apo (black), cAMP (red) and cGMP (green) conditions. (C) Spaghetti plot of distance distributions for each experiment (n=4) (thin lines). For comparison, chiLife distributions are overlayed (dashed curves). (D) Summary of Gaussian fit standard deviations, σ, versus average distances, r̄, for apo and cAMP, with average values as cross marks. Colors correspond to conditions in (A-C). (E) Fit values and averages for fraction activation (A2) for each condition.

Calculated Energetics of the Four State Model

Lifetime measurements in 150 mM KCl versus 500 mM KCl conditions. (A) Normalized SEC traces of SthKC-term in 150 mM KCl (solid curve) and 500 mM KCl (dashed curve). (B) Representative Weber plot for [Fe(phenM)3]2+ lifetimes in 150 mM (open circles) and 500 mM KCl (closed triangles), in each ligand condition (apo, black; cGMP, green; cAMP; red). An additional lifetime trace is shown for an experiment where additional 300 mM KCl was added to the protein sample with 150 mM KCl in cGMP (closed green circles). (C) Comparison of Gaussian average distance, r̄, and standard deviation, σ, between ionic concentrations using model global fitting of [Fe(phenM)3]2+ and [Ru(bpy)2phenM]2+ with the sum of two Gaussians (apo, black, and cAMP, red, n=4). (D) Fraction of active state in each condition (A2) for 150 mM KCl and 500 mM KCl using the sum of two Gaussian model fits [apo P=0.2, cGMP *P=0.0001].

Steady-state tmFRET of monomeric and tetrameric SthKC-term. (A) SEC chromatogram showing separation of monomeric (empty triangle, blue area) and tetrameric protein (solid triangle, gray area) fractions for SthKC-term-I359Acd and SthKC-term-I359Acd-R417C (no WT SthKC-term present) on a Superdex 75 increase 10/300 column (GE Healthcare). (B) Structure showing SthKC-term-I359Acd with [Cu(TETAC)]2+ acceptor at R417C (PDB:4D7T). (C) Fluorescence time-course of SthKC-term-I359Acd-417C upon addition of [Cu(TETAC)]2+, cAMP and TCEP in tetrameric protein (closed diamonds) and monomeric protein (open triangles). (D) Summary of FRET efficiencies for monomer and tetramer, with mean values shown as horizontal lines. Left: SthKC-term-I359Acd-R417C-[Cu(TETAC)]2+ (apo, *P=0.0005, cAMP, P=0.06). Right: SthKC-term-S361Acd-V416C-[Fe(phenM)3]2+ apo, *P=0.006, cAMP: P=0.9).

Representative Weber plot of measured lifetimes of SthKC-term-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.

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 (τDl, τD2) (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 (r̅l and r̅2), standard deviations (σl and σ2) and relative amplitude of the second component (A2). The donor-only fraction (fD) 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 (Dω) and out-of-phase (Nω) components are plotted for the measured, corrected, and background fluorescence signal as a function of the excitation modulation frequency (ω), where fB is the fraction of the fluorescence intensity due to the background. (E) Plot of the phase delay (φω) and modulation ratio (mω) 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.

Representative phasor plot of measured lifetimes of SthKC-term-S361Acd-V416C alone (donor-only, grey), in the presence of [Ru(bpy)2phenM]2+ (apo, black), in the presence of 1 µM cAMP (cyan) and presence of 1.23 mM cAMP (red).

Identifiability of parameters in the sum of two Gaussian distributions model, with global fitting Fe(phenM)3]2+ and [Ru(bpy)2phenM]2+ data. (A-B) Minimization of χ2 values for the Gaussian average distance (r̄) (A) and standard deviation (σ) (B) for the resting (black) and active states (red). (C) Minimization of χ2 values for parameter A2 for the conditions of apo, cAMP and cGMP. (D) Minimization of χ2 values for the fraction of donor-only component (fD) in protein samples for each experiment of apo and cAMP for [Fe(phenM)3]2+, of apo and cAMP for [Ru(bpy)2phenM]2+, of cGMP for [Fe(phenM)3]2+, and of cGMP for [Ru(bpy)2phenM]2+.