AP09 Cordes: Single-molecule studies of conformational switching and activation of the sodium-coupled symporter BetP
The sodium-coupled betaine symporter BetP from Corynebacterium glutamicum counteracts hyperosmotic stress and is regulated via an osmosensing C-terminal domain. While the biochemistry of the system is well-characterized, structural information is only partially available due to a lacking C-terminal domain in the crystal structure. In this project, we develop novel biophysical methods for studies of structural dynamics in the transporter. For this we will establish site-specific fluorescence labelling of the homotrimeric active transporter with two fluorescent probes enabling Förster-resonance energy transfer (FRET) experiments. Our approach is based on two distinct experimental strategies using (i) heterotrimic expression of the transporter and (ii) photochemical caging of fluorescent dyes for labelling of the homotrimer. With this technology we plan to characterize both the catalytic action of the transporter and the conformational switching of the C-terminal regulatory domain required for transport. In detail we will assess potassium- and ligand-induced conformational changes of the transmembrane and C-terminal domains by employing FRET in combination with single-molecule detection. In preliminary experiments, we observed major differences in the conformational states in polymeric amphipols compared to detergent environment of the C-terminal domain. Strikingly, the observed differences and specifically the state identified in amphipole likely reflects the native cellular state of the C-terminal domain of BetP. Finally, we investigated the effect of potassium and osmo-activated conditions on the conformational states of BetP. We demonstrate that BetP senses and rearranges the C-terminal helices in response to increasing concentration of K+ within an optimal protein/Amphipol complex that represents the native protein state. This helical conformation is likely to differ from the X-ray crystal structure of BetP that was previously obtained in detergent environment. Our preliminary findings ascribe a new role to potassium by promoting conformational switching which is relevant for the BetP activation cycle to promote the substrate-bound closed state.