EmrE is a multidrug resistance efflux pump with specificity to a wide range of antibiotics and antiseptics. To obtain atomic-scale insight into the attributes of the native state that encodes the broad specificity, we used a hybrid of solution and solid-state NMR methods in lipid bilayers and bicelles. Our results indicate that the native EmrE dimer oscillates between inward and outward facing structural conformations at an exchange rate (kex) of ∼300 s–1 at 37 °C (millisecond motions), which is ∼50-fold faster relative to the tetraphenylphosphonium (TPP+) substrate-bound form of the protein. These observables provide quantitative evidence that the rate-limiting step in the TPP+ transport cycle is not the outward–inward conformational change in the absence of drug. In addition, using differential scanning calorimetry, we found that the width of the gel-to-liquid crystalline phase transition was 2 °C broader in the absence of the TPP+ substrate versus its presence, which suggested that changes in transporter dynamics can impact the phase properties of the membrane. Interestingly, experiments with cross-linked EmrE showed that the millisecond inward-open to outward-open dynamics was not the culprit of the broadening. Instead, the calorimetry and NMR data supported the conclusion that faster time scale structural dynamics (nanosecond–microsecond) were the source and therefore impart the conformationally plastic character of native EmrE capable of binding structurally diverse substrates. These findings provide a clear example how differences in membrane protein transporter structural dynamics between drug-free and bound states can have a direct impact on the physical properties of the lipid bilayer in an allosteric fashion.

Secondary active transport proteins play a central role in conferring bacterial multidrug resistance. In this work, we investigated the proton-coupled transport mechanism for the Escherichia coli drug efflux pump EmrE using nuclear magnetic resonance (NMR) spectroscopy. Our results show that the global conformational motions necessary for transport are modulated in an allosteric fashion by the protonation state of a membrane-embedded glutamate residue. These observations directly correlate with the resistance phenotype for EmrE and the E14D mutant as a function of pH. Furthermore, our results support a model in which the pH gradient across the inner membrane of E. coli may be used on a mechanistic level to shift the equilibrium of the transporter in favor of an inward-open resting conformation poised for drug binding.

We used oriented solid-state NMR spectroscopy and biochemical cross-linking experiments to demonstrate that the ligand-free membrane protein transporter EmrE forms anti-parallel dimers with different monomer tilt angles relative to the lipid bilayer. Our results also show the subtle conformational changes efflux pumps experience in response to drug binding and emphasize the importance of studying membrane proteins in a fluid bilayer environment.

Quantitative evaluation of small molecule permeation and accumulation in Gram-negative bacteria is important for drug development against these bacteria. While these measurements are commonly performed at physiological pH, Escherichia coli and many other Enterobacteriaceae infect human gastrointestinal and urinary tracts, where they encounter different pH conditions. To understand how external pH affects permeation and accumulation of small molecules in E. coli cells, we apply second harmonic generation (SHG) spectroscopy using SHG-active antimicrobial compound malachite green as the probe molecule. Using SHG, we quantify periplasmic and cytoplasmic accumulations separately in live E. coli cells, which was never done before. Compartment-wise measurements reveal accumulation of the probe molecule in cytoplasm at physiological and alkaline pH, while entrapment in periplasm at weakly acidic pH and retention in external solution at highly acidic pH. Behind such disparity in localizations, up to 2 orders of magnitude reduction in permeability across the inner membrane at weakly acidic pH and outer membrane at highly acidic pH are found to play key roles. Our results unequivocally demonstrate the control of external pH over entry and compartment-wise distribution of small molecules in E. coli cells, which is a vital information and should be taken into account in antibiotic screening against E. coli and other Enterobacteriaceae members. In addition, our results demonstrate the ability of malachite green as an excellent SHG-indicator of changes of individual cell membrane and periplasm properties of live E. coli cells in response to external pH change from acidic to alkaline. This finding, too, has great importance, as there is barely any other molecular probe that can provide similar information.

Substance P (SP) is one of the target neurotransmitters associated with diseases related to chronic inflammation, pain and depression. The selective receptor for SP, NK(1)R is located in the heterogeneous microdomains or caveolae in membrane. Gangliosides, specifically GM1, are markers of these heterogeneous sites. Also, gangliosides are considered as important regulatory elements in cell-cell recognition and cell signaling. In the present work, we describe the conformations of Substance P in the presence of ternary membrane systems containing GM1 at the physiological concentration. SP is mostly unstructured in water, but appears as extended 3(10) helical or turn III in isotropic bicelles, more pronounced in the presence of GM1. NMR results suggest that, in the GM1 containing bicelles, the peptide is more inserted into the membrane with its C-terminus, while N-terminus lies close to the membrane-water interface. The NMR-derived conformation of SP in GM1 bicelles is docked on homology modeled NK(1)R and resulting interactions satisfy reported mutagenesis, fluorescence, photo-affinity labeling and modeling data. The results highlight efficacy of GM1 in membrane in providing structure in an otherwise flexible neurotransmitter Substance P; thus providing indication that it may be useful also for other neurotransmitter peptides/proteins associated with membrane.