Fig. 9.

Proposed future single transporter recordings using periplasmic substrate binding protein-based fluorescent biosensors. A, Schematic of the proposed experimental set-up for single-transporter recordings. Single-transporters are reconstituted into proteoliposomes at low protein-to-lipid ratios to have one (or none) transporter per liposome, providing positive and negative control experiments. The position of the liposome can be detected via a fluorescent marker. Liposomes are filled with a large quantity of fluorescent sensors such as SBPs that detect changes in the concentration of the substrate, here via increase in their signal. B, Assay design for use of MalE as maltose-sensor using an environmentally-sensitive fluorophore (blue: IANBD). Fluorescence spectra at increasing maltose concentration from 0 (low fluorescence) to 1 mM maltose (high fluorescence). C, Scheme of the TIRF-microscope with laser box and dual-view allowing simultaneous recording of two emission channels, exemplified by data from a lipid marker DiD and luminal dsDNA-fluorescein. D, Example data of fluorescent markers in lumen and the liposome leaflet as schematics for expected signals upon single-transporter recordings. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)