BN seminar - Thorben Cordes (University of Groningen): "Single-molecule tools for the study of membrane transporters"





Room F, Delft


In the first part of my talk I focus on mechanistic studies of ABC importers, which play a pivotal role in the physiology of microorganisms. To decipher how conformational changes within the subdomains drive the overall membrane transport, we focus on the homodimeric GlnPQ complex, possessing two different substrate-binding proteins (SBDs) per single translocator. Based on single-molecule FRET, ITC and uptake assays, we show that both SBDs have evolved to capture different amino acids by an undocumented type of induced-fit mechanism: they have the capability to intrinsically close in the absence of a ligand. While the presence of high-affinity ligands leads to increased transitions of the SBDs into the closed conformation at constant closed state lifetime, the presence of the low-affinity ligands shortens the closed state lifetime by two orders of magnitude and enhances the overall transport rate. Engineering the ligand’s affinity and hence the close state’s lifetime of each SBD allows us to tune the transport activity. These findings indicate that the intrinsic closing regulates SBD docking to the translocator and following-up transport.
Secondly, I describe our attempts to create "self-healing" fluorophores with automatic repair of triplet-induced photodamage for applications such as smFRET. I present an improved strategy to scaffold self-healing fluorophore with the aim to further increase their photostability. We present results for self-healing fluorophores of various dye classes including applications in demanding experiments such as STED-nanoscopy.