Fig. 1.

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, $K_{1/2}$: 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 $\text{ΔG}$’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, $\overline{r}$, heterogeneity within each conformational state as standard deviation, $\sigma$, and heterogeneity between conformational states as fractional area $A_2$ for apo (black) and holo (red) conditions.