An electrode surface is presented that enables the characterisation of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo3 (cbo3), has been co-immobilised into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalised with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self assembly of proteoliposomes and its structure is characterised by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS) and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo3 is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo3 is mediated by the membrane-localised ubiquinol-8, the physiological electron donor of cbo3. Enzyme coverages observed with TM-AFM and CV coincide (2–8.5 fmol·cm−2) indicating that most - if not all - cbo3 on the surface is catalytically active and thus retains its integrity during immobilisation.

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport (Cloherty, E. K., Levine, K. B., & Carruthers, A. (2001). The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry, 40(51), 15549–15561). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but non-competitively inhibits sugar uptake into cells lacking sugar at 4°C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [3H]-CB binding to red cell membranes depleted of peripheral proteins at 4°C. Others produce a moderate stimulation of [3H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogs modestly stimulate [3H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure and we evaluate the major determinants of ligand binding affinity and cooperativity.

Mhp1, a hydantoin transporter from M. liquefaciens, was purified and crystallized. Diffraction data were collected to 2.85 Å resolution; the crystal belonged to the orthorhombic space group P212121.

The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8 Å). A complete data set has been collected from a single crystal to a resolution of 2.85 Å with 64 741 independent observations (94% complete) and an R merge of 0.12. Further experimental phasing methods are under way.

Crystal structures of a membrane protein transporter in three different conformational states provide insights into the transport mechanism.

Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.

The structure of the sodium-benzylhydantoin transport protein, Mhp1, from Microbacterium liquefaciens comprises a 5-helix inverted repeat, which is widespread amongst secondary transporters. Here we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 Å resolution, complementing its previously-described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward- facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8 and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His6 tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent Km values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the l isomer over the d isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of β-glucuronides with synthetic [14C]phenyl-1-thio-β-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of β-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.

The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.