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. 2015 Oct 28;112(45):13874–13879. doi: 10.1073/pnas.1512994112

Fig. S1.

Fig. S1.

Use of Trp-IAEDANS FRET as a reporter of LacY topological rearrangement. (A) Trp fluorescence of LacY containing a diagnostic Trp replacement in EMD C6 observed with unlabeled H205W/V331C mutant. AU, arbitrary units. Spectra were recorded with excitation at 295 nm for 1 μM protein in 10 mM Tris⋅HCl (pH 7.5). An increase in Trp fluorescence as a function of PE content is indicated by the upward black arrow. (B) Quenching of Trp fluorescence (indicated by the black arrow) in the C6 EMD of LacY and appearance of Trp-IAEDANS FRET (indicated by the blue arrow) observed with IAEDANS-labeled H205W/V331C mutant. Spectra were recorded with excitation at 295 nm for 1 μM protein in 10 mM Tris⋅HCl (pH 7.5). (C) Fluorescence of LacY containing a diagnostic Trp replacement in EMD P7 observed with IAEDANS-labeled F250W/V331C mutant. Spectra were recorded with excitation at 295 nm for 1 μM protein in 10 mM Tris⋅HCl (pH 7.5). (D) Fluorescence of LacY containing only its six native Trp residues with IAEDANS-labeled V331C mutant. Spectra were recorded with excitation at 295 nm for 1 μM protein in 10 mM Tris⋅HCl (pH 7.5). (E) FRET efficiency as a function of the amount of PE present in LacY proteoliposomes. FRET efficiency (E) was calculated from steady-state fluorescence spectra as E = 1 − (FDA/FD), where FDA and FD are the donor fluorescence intensities at 340 nm in the presence and absence of the acceptor, respectively. Experiments were performed in triplicate, and error bars indicate SD (black, Trp in C6; gray, Trp in NT). (F) Real-time measurements of Trp fluorescence (Upper) and IAEDANS fluorescence (Lower) after excitation at 295 nm. (Upper) Red (C6) and blue (NT) traces indicate Trp fluorescence observed with unlabeled H205W/V331C and L14W/V331C mutants, respectively, upon addition of PE; the black trace indicates Trp fluorescence observed with unlabeled H205W/V331C in the absence of lipid exchange; and magenta and orange traces indicate Trp fluorescence observed with unlabeled V331C containing only endogenous Trp (no Trp replacement in EMDs) upon addition and dilution of PE, respectively. (Lower) Red (C6) and blue (NT) traces indicate FRET observed with IAEDANS-labeled H205W/V331C and L14W/V331C mutants, respectively, upon addition of PE, and the black trace indicates FRET observed with IAEDANS-labeled H205W/V331C in the absence of lipid exchange.