Figure 4.

Real-time determination of the reversibility of phosphorylation-induced topological switch of a membrane protein upon dephosphorylation in proteoliposomes. A, real-time FRET measurements monitoring the topological switch of EMD C6 in LacY in the presence of encapsulated phosphatase with or without addition of kinase inhibitor (Inhib) GSK 2334470. LacY template −2/0/−2 containing a single engineered PDK1 sequence in EMD C6 was reconstituted in proteoliposomes made of E. coli native lipids. Data represent the averaged normalized fluorescence expressed as the ratio F_Meas/F₀. Experiments were repeated three to five times, and the data represent mean values. B, schematics of the measured events, with the inhibited steps (phosphorylation and topological inversion) shaded.