Fig. 4.

The potassium-dependent isotropic chemical shifts of E71 and D80 are shown. (A) The ¹³C chemical shifts of E71 (Top) and D80 (Bottom) in WT-KcsA are plotted as a function of ambient [K⁺] at pH 7.5. The data show a large downfield movement in the side-chain carboxyl chemical shift of E71 (in yellow) as [K⁺] is increased, but no change in the backbone shifts. At [K⁺] = 10 μM, both states of E71 are observed, indicated by the filled and hollow markers. The D80 isotropic shifts remain unchanged by potassium. (B) Shows ¹³C–¹³C correlation spectra highlighting the E71 and D80 carboxyl chemical shifts at the endpoints of the titration. (C) The relative population of the deprotonated state of E71 based on its NMR signal intensity is overlaid with populations from another selectivity filter marker, V76. The V76 curve (fit to a two-state binding model) shows that the transition from the conductive state to the collapsed state occurs between [K⁺] = 1 μM and 10 μM. The data show that change in the E71 side chain occurs in the same potassium-concentration regime as the structural collapse of the filter based on K_d measurements of selectivity filter markers. Therefore these two processes are very likely coupled.