No description available.

No description available.

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.

No description available.

The Major Intrinsic Proteins are represented in all forms of life; plants, animals, bacteria and recently archaebacteria have all been shown to express at least one member of this superfamily of integral membrane proteins. We have overexpressed the E. coli aquaglyceroporin, glycerol facilitator (GlpF), to use as a model for studying membrane protein structure, folding and stability. Understanding membrane protein folding, stability, and dynamics is required for a molecular explanation of membrane protein function and for the development of interventions for the hundreds of membrane protein folding diseases. X-ray analysis of GlpF crystals shows that the protein exits as a tetramer in the crystallized state [1]. However, preparations of stable aqueous detergent solutions of GlpF in its native oligomeric state have been difficult to make; the protein readily unfolds and forms non-specific aggregates in many detergents. Here, I report the study of the structure and stability of the glycerol facilitator in several detergent solutions by blue native and sodium dodecyl sulphate polyacrylamide gel electrophoresis, circular dichroism, and fluorescence. For the first time, stable protein tetramers were prepared in two different detergent solutions (dodecyl maltoside (DDM) and lyso-myristoyl phosphatidylcholine (LMPC)) at neutral pH. Thermal unfolding experiments show that the protein is slightly more stable in LMPC than in DDM and that the thermal stability of the helical core at 95oC is slightly greater in the former detergent. In addition, tertiary structure unfolds before quaternary and secondary structures in LMPC whereas unfolding is more cooperative in DDM. The high stability of the protein is also evident from the unfolding half-life of 8 days in 8 M urea suggesting that hydrophobic interactions contribute to the stability. The GlpF tetramers are less resistant to acidic conditions; LMPC-solubilized GlpF shows loss of tertiary and quaternary structure by pH 6, while in DDM the tertiary structure is lost by pH 5, however the tetramer remains mostly intact at pH 4. The implications of thermal and chemical stress on the stability of the detergent-solubilized protein and its in vivo folding are discussed.

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.

No description available.

No description available.

Arguably the greatest confrontation for humanity and the Arguably the greatest confrontation for humanity and the natural world is addressing the shortfalls of our current energy sources and the threat that intensive consumption has on the environment. By harvesting the immense energy of the sun and converting it into clean fuels such as H2, these issues may be resolved. Inefficiencies of current technology have made the realization of an energy changeover extremely challenging. A viable solution would be an integrated system of the absorbing and conversion components embedded in a membrane. The membrane must house the electrode assembly, block product crossover, and manage ionic and electronic charges generated while remaining passive to the photoelectrochemical process. The first approach to developing such a membrane involves the formation of a composite of the electrically conducting polymer PEDOT and the inorganic acid PMA. It was found that the material possessed excellent electrical conductivity as a function of pH and oxidation state, and stability against overoxidation, while the ionic conductivity remained insufficient. This was combatted with the addition of the proton conductor Nafion®, which was combined in the desired ratio to optimize the material conductivities. Membranes capable of maintaining steady-state pH gradients, with the motivation to operate the oxygen- and hydrogen-generating sides in their optimal pH, were also investigated. It is herein confirmed that these membranes are able to maintain a pH gradient of 14 units indefinitely while adding no additional thermodynamic perturbance to the system. Membranes were constructed by combining ion exchange layers with interchangeable materials in an interfacial layer to develop a photoelectrochemically-adapted membrane. A transparent conducting oxide, conducting polymer, and graphene materials were selected, with the former two exhibiting inadequate activity. However, it was found that graphene oxide demonstrates activity that is comparable or better than commercially available membranes. Its presence also stabilized the membrane. The shortfall of graphene oxide is that it is an insulator. Chemical reduction was used to introduce electrical conductivity by removing the functional groups, which was controlled by the exposure conditions. It is shown that a reduced graphene oxide membrane can meet the figures of merit outlined for these integrated energy systems.

KefC is a potassium transport system of <i>E.coli</i> that is regulated by glutathione metabolites. An analysis of the KefC protein was undertaken in order to advance towards an understanding of the transport and regulatory processes at a molecular level. KefC-LacZ hybrid proteins were constructed by mini-Mu transposon insertion mutagenesis into <i>kefC</i> plasmids. The distribution of the B-galactosidase activity between membrane and soluble fractions indicated that the KefC moieties of the hybrid proteins were directing the proteins to the membrane, suggesting that KefC is a membrane protein. Sequencing the fusion junctions of the <i>kefC'-'lacZ</i> gene fusions allowed progress to be made towards topological mapping of the KefC protein. Two stable, high activity hybrid proteins confirmed the location of the first cytoplasmic loop and the large, cytoplasmic C-terminal domain proposed on the basis of the deduced amino acid sequence. A knowledge of the gene orientation, derived from restriction mapping of the transposon insertions, enabled kefC to be cloned downstream of a bacteriophage T7 promoter and expressed using the T7 polymerase/promoter system, overcoming initial problems of low expression. This identified KefC as a membrane located protein of apparent molecular mass 55-60 kDa. The oligomerization of KefC was investigated. It was shown that treatment with the cross-linking reagent formaldehyde moved the KefC band to a higher molecular weight and it was suggested that KefC-LacZ hybrid proteins interfered with potassium efflux via KefC. These observations are consistent with KefC functioning as an oligomer. Regimes suitable for solubilization and purification of KefC-LacZ hybrid proteins were developed. Attempts were made to generate antibodies against KefC but encountered difficulties due to contamination with antibodies not specific for KefC.