Single-pair FRET experiment of membrane protein insertion in solution.

(A) Donor-labeled Pf3 coat protein was added to proteoliposomes containing acceptor-labeled YidC protein and fluorescence was followed in the confocal volume. (B) Positions of the donor fluorophor (Atto520, blue dots) at residues 16 and 48 in the Pf3 coat protein and the acceptor fluorophor (Atto647N, red dots) at periplasmic (23, 442, 511) and cytoplasmic (7, 405, 478) residues of YidC. (C) Complementation assay of the YidC single-cysteine mutants. Growth under glucose conditions indicates functional complementation. Asteriks denote added 1 mM IPTG in the glucose containing LB agar to optimize expression.