Figure 6.

Insertion of single protein molecules into YidC proteoliposomes observed by FCS-based FRET. Atto520-labeled Pf3 protein was added to a solution containing Atto647N-labeled YidC, reconstituted into proteoliposomes. (A) The high temporal resolution of distance measurements between fluorophores, determined by FRET, allows resolving the individual events taking place on a molecular level, as illustrated in (B). The protein Pf3 (red) freely diffuses in solution (a), until it encounters a YidC-containing proteoliposome and binds to the insertase (green, b). The distance between the fluorophores decreases, which demonstrates a close interaction between YidC and Pf3 during the membrane insertion process (c). The increasing distance between donor and acceptor fluorophor and finally the loss of a FRET signal indicate the release of Pf3 from the insertase YidC (d, adapted from (13)). To see this figure in color, go online.