Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.

BetP, a trimeric Na þ -coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K þ -concentration. This transport regulation of BetP depends on a sophisticated interaction network. Using single-molecule force spectroscopy we structurally localize and quantify these interactions changing on K þ -dependent transport activation and substrate-binding. K þ significantly strengthened all interactions, modulated lifetimes of functionally important structural regions, and increased the mechanical rigidity of the symporter. Substrate-binding could modulate, but not establish most of these K þ -dependent interactions. A pronounced effect triggered by K þ was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K þ -concentrations akin to those BetP encounters during hyperosmotic stress trigger the formation of a periplasmic second betaine-binding (S2) site, which was found to be at a similar position reported previously for the BetP homologue CaiT. In BetP, the presence of the S2 site strengthened the interaction between EH2, transmembrane α-helix 12 and the K þ -sensing C-terminal domain resulting in a K þ -dependent cooperative betaine-binding.atomic force microscopy | energy landscape | membrane transporter | osmoregulation | secondary substrate-binding site

The opportunistic pathogen Acinetobacter baumannii is able to grow on carnitine. The genes encoding the pathway for carnitine degradation to the intermediate malic acid are known but the transporter mediating carnitine uptake remained to be identified. The open reading frame HMPREF0010_01347 ( aci01347) of Acinetobacter baumannii is annotated as a gene encoding a potential transporter of the betaine/choline/carnitine transporter (BCCT) family. To study the physiological function of Aci01347, the gene was deleted from A. baumannii ATCC 19606. The mutant was no longer able to grow on carnitine as sole carbon and energy source demonstrating the importance of this transporter for carnitine metabolism. Aci01347 was produced in Escherichia coli MKH13, a strain devoid of any compatible solute transporter, and the recombinant E. coli MKH13 strain was found to take up carnitine in an energy‐dependent fashion. Aci01347 also transported choline, a compound known to be accumulated under osmotic stress. Choline transport was osmolarity‐independent which is consistent with the absence of an extended C‐terminus found in osmo‐activated BCCT. We propose that the Aci01347 is the carnitine transporter mediating the first step in the growth of A. baumannii on carnitine.

Members of the genus Acinetobacter are well known for their metabolic versatility that allows them to adapt to different ecological niches. In previous studies, we have demonstrated that Acinetobacter baylyi ADP1 can cope with high salinities by uptake and accumulation of the well-known compatible solute glycine betaine. Here, we demonstrate that addition of choline restores growth at high salinities. We further show that choline was actively taken up by the cells and converted to glycine betaine. Uptake of choline was induced by high salinity and the presence of choline in the growth medium. At high salinities, glycine betaine was accumulated in the cells whereas in the absence of osmotic stress it was exported. Inspection of the genome sequence followed by mutant studies led to the identification of two genes encoding secondary transporters (BetT1 and BetT2) of the betaine-choline-carnitine transporter (BCCT) family. The BetT1 transporter lacks an extended C-terminal domain usually found in osmoregulated choline BCCTs. BetT1 was found to facilitate osmolarity-independent choline transport most likely by a uniport mechanism. We propose that BetT1 does not primarily function in osmoadaptation but might play a role in metabolic adaptation to choline-rich environments.

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor–acceptor pairs during solution-based smFRET. We use this “caged FRET” methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.