Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report 13 Multidrug drug resistance, in particular bacterial resistance to clinical antibiotics, is a widely known phenomenon. Basic defense mechanisms of bacteria include permeability barriers, inactivation of antimicrobials, modification of antibiotic targets, and active drug efflux (1). Active efflux is conducted by primary and secondary active transport proteins and the latter are found in almost all transporter families (2). The molecular mechanism of the broad substrate specificity of multidrug efflux pumps is not yet fully understood. To this end structural studies are desirable, but currently only 13 three-dimensional structures of different transport proteins are known and not every transport family is represented. The only multidrug transporter with known three-dimensional structure is AcrB (4, 5). A three-dimensional structure of the ABC transporter Sav1866 has also been reported (6), which is assumed to function as a multidrug efflux pump.Here, we are especially interested in Escherichia coli EmrE, a member of the medically relevant SMR 2 protein family (TC number 2.A.7.1 (8, 9)). Due to its small size (12 kDa), EmrE was originally proposed as ideal structure-function paradigm (10). It has attracted significant interest due to its controversial topological organization (11-15), oligomerization state (16 -19), transport cycle steps (12, 20 -23), and unknown three-dimensional structure (24). EmrE transports a diverse array of aromatic, positively charged substrates in exchange for protons (21) via at least one occluded transport cycle intermediate state (25). Other SMR proteins have overlapping but significantly different substrate specificities with measured affinities in the nanomolar to millimolar range (20, 26). All SMR proteins are of similar size (ϳ11-12 kDa), have a 4-transmembrane helix topology and a highly conserved key residue Glu 14 (20,27). It has been shown that Glu 14 is an essential residue and directly involved in drug and proton binding (28 -30). It can reasonably be assumed, that Glu 14 of both protomers in a dimer form a shared binding pocket (31,32).Whether EmrE forms a symmetric or an asymmetric dimer should be reflected in the chemical shifts of residues such as Glu 14 , which are likely to be found at the dimerization interface. We therefore 13 C-labeled Glu 14 in EmrE by utilizing a cell-free expression system. To allow an unambiguous NMR analysis, the nonessential residue Glu 25 was replaced with alanine (EmrE E25A) to create a single glutamate mutant. The protein was reconstituted into E. coli lipids allowing the most native environment possible. The sample was maintained at pH 8.0. Under these conditions, withou...

A highly versatile water-soluble pyridine-spiropyran photoswitch is reported which functions as photoacid in a wide pH range. Under neutral conditions, the open-ring merocyanine (MC) exists to 48% and closes quantitatively by irradiation with visible light, while the reverse reaction occurs rapidly in the dark or by irradiation at 340 nm. The different pK of the pyridine nitrogen in the closed spiropyran (4.8) and open merocyanine form (6.8) leads to a reversible proton release in a pH range of 3-7. Only negligible hydrolytic decomposition was observed in the pH range from 1 to 12. The application of potentially harmful UV light can be circumvented due to the fast thermal ring-opening except for pH values below 3. Its photoacidic properties make this compound an effective pH-regulating photoswitch in water and enable controlled proton-transfer processes for diverse applications. Additionally, all of the involved protonated states of the compound exhibit discriminative fluorescence features within certain pH ranges, which even expands its utility to a light-controllable, pH-sensitive fluorophore.

Numerous studies in biological and material sciences have used nitro-BIPS, dinitro-BIPS as well as Py-BIPS as versatile photoswitches. Still, the photochemical picture of this class of compounds is far from complete. We present photometric steady-state and ultrafast time-resolved pump/probe spectroscopic measurements on water-soluble derivatives of these three spriopyrans. Our experiments reveal significant differences between the nitro-substituted spiropyrans and Py-BIPS. In contrast to the high resistance of Py-BIPS towards hydrolysis over weeks, the two nitro-BIPS derivatives decompose over hours. The fluorescence properties of Py-BIPS are unique in showing an emission of the spiro photoisomer. The ringopening and -closing reaction of Py-BIPS is accomplished within picoseconds, whereas nitro-derivatives photoisomerize on longer time scales. These long-lived transients indicate either the contribution of triplet states or the involvement of multiple merocyanine isomers in the reaction pathway. Scheme 1. Reversible photochromic reaction between the closed spiropyran (SP) and the open-ring merocyanine (MC, TTC comformation) isomer of the BIPS compound (top and middle). The three water-soluble spiropyran derivatives (1-3) investigated in this study (bottom)..

Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand ‘azoCm’ was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a K D of 545 nM. As the aptamer cannot bind to the irradiated ligand ( λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.

Photoacids attract increasing scientific attention, as they are valuable tools to spatiotemporally control protonrelease reactions and pH values of solutions. We present the first time-resolved spectroscopic study of the excited state and proton-release dynamics of prominent merocyanine representatives. Femtosecond transient absorption measurements of a pyridine merocyanine with two distinct protonation sites revealed dissimilar proton-release mechanisms: one site acts as a photoacid generator as its pK a value is modulated in the ground state after photoisomerization, while the other functions as an excited state photoacid which releases its proton within 1.1 ps. With a pK a drop of 8.7 units to À 5.5 upon excitation, the latter phenolic site is regarded a super-photoacid. The 6-nitro derivative exhibits only a phenolic site with similar, yet slightly less photoacidic characteristics and both compounds transfer their proton to methanol and ethanol. In contrast, for the related 6,8-dinitro compound an intramolecular proton transfer to the orthonitro group is suggested that is involved in a rapid relaxation into the ground state.