Fig. 4.

The oligomeric states of the isolated RCK protein depend on Ca²⁺ and pH. (A) Size determinations in different pH solutions in the presence of 5 mM Ca²⁺ (blue) or EGTA (red) using size-exclusion HPLC (smooth lines, 280 nm absorption) and static light scattering (dots, the molecular masses on the y axes). Arrowheads indicate the molecular mass of each oligomeric state calculated from the amino acid sequence. (B) Sedimentation equilibrium analysis of the isolated RCK domain in pH 8.5 EGTA solution reveals that the domain is at equilibrium between monomer and hexamer, which also match the sizes derived from static light scattering measurement. (C) Summary of the oligomeric states of the isolated RCK protein in different pHs in the presence (blue) or absence (red) of Ca²⁺. (D) Fluorescence intensity of Trp-123 changes over time upon Ca²⁺ binding to the isolated RCK protein. The fluorescence measurement is indicated with gray dots. Single-exponential (tau = 5.7 sec) and double-exponential fits are shown in blue and red, respectively.