The biological membrane is composed of lipids and proteins that make up dynamic barriers around cells and organelles. Membrane-spanning proteins are involved in many key processes in the cell such as energy conversion, nerve conduction and signal transduction. These proteins interact closely with lipids as well as with other proteins in the membrane, which modulates and affects their structure and function. In the energy-conversion process, membrane-bound proton-transport proteins maintain an electrochemical proton gradient across the mitochondrial inner membrane or the cytoplasmic membrane of bacteria. This gradient is utilized for ATP synthesis or transport of ions and molecules across the membrane. Results from earlier studies have shown that proton transporters are influenced by their environment. Here, one of these proton transporters, cytochrome c oxidase, has been purified and reconstituted into liposomes or nanodiscs and membrane effects on specific proton-transfer processes were studied. In these studies we observed that the membrane accelerated proton transfer to the surface of cytochrome c oxidase and that there is a protonic link, via a Glu residue that mediates proton transfer from the membrane surface to a proton-transfer pathway in this protein. In addition, the membrane was shown to modulate specific internal electron and proton-transfer reactions. The results from these studies show that the membrane composition influences transmembrane transport. Consequently, our understanding of these processes requires investigation of these transporter proteins in different membrane-mimetic systems of variable and well-defined composition. Furthermore, the data show that membrane surfaces facilitate lateral proton transfer which is presumably essential for maintaining high efficiency in energy conversion. This is particular important in organisms such as alkaliphilic bacteria where the driving force of the electrochemical proton gradient, between the bulk solution on each side of the membrane is not sufficient for ATP synthesis.

There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures. Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.

α-helical membrane proteins comprise about a quarter of all proteins in a cell and carry out a wide variety of essential cellular functions. This thesis is focused on topology analyses of bacterial membrane proteins. The topology describes the two-dimensional structural arrangement of a protein relative to the membrane. By combining large-scale experimental and bioinformatics techniques we have produced experimentally constrained topology models for the major part of the Escherichia coli membrane proteome. This represents a substantial increase in available topology information for bacterial membrane proteins. Many membrane protein structures show signs of internal duplication and approximate two-fold in-plane symmetry. We propose a step-wise pathway to explain how proteins with such internal inverted repeats have evolved. The pathway is based on the ‘positive-inside’ rule and starts with a protein that can adopt two topologies in the membrane, i.e. a “dual” topology protein. The gene encoding the dual topology protein is duplicated and eventually, through re-distribution of positively charge residues, the two resulting homologous proteins become fixed in opposite orientations in the membrane. Finally, the two proteins may fuse into one single polypeptide with an internal inverted repeat structure. Finally, we re-create the proposed step-wise evolutionary pathway in the laboratory by showing that only a small number of mutations are required in order to transform the homo-dimeric, dual topology protein EmrE into a hetero-dimeric complex composed of two oppositely oriented proteins.

Membrane proteins are key players in many biological processes. Since most membrane proteins are assembled into oligomeric complexes it is important to understand how they interact with each other. Unfortunately however, the assembly process (i.e. their social life) remains poorly understood. In the work presented in this thesis I have investigated when and how membrane proteins assemble with each other and their cofactors to form functional units. We have shown that that cofactor insertion in the hetero-tetrameric cytochrome bo3 occurs at an early state in the assembly process. We also found that the pentameric CorA magnesium ion channel is stabilised by different interactions depending on the magnesium ion concentration in the cell. These studies indicate that the assembly of a functional unit is a dynamic process, which is a result of many different forces. By studying the assembly of membrane proteins we have obtained a deeper insight into their function, which cannot be explained by static crystal structures. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

No description available.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

The bath-to-cell transport, cytosolic concentration, and tubular content of methylmercury (Me203Hg+) and the sulfhydryl-amino acids and sulfhydryl-amino acid derivatives conjugated to Me203Hg+ were studied in the non-perfused S2 segments of the proximal tubule of the rabbit kidney. Active transport of Me203Hg+ was established by a temperature dependent (greater than 100% reduction in bath-to-cell transport, 99% decrease in cytosolic concentration, 63% decline in the tubular contents at 12°C when compared to 37°C). Conjugates of Me203Hg+ showed mixed results, with L-cysteine and L-taurine demonstrating the most significant increase in uptake. Transport of Me203Hg+-L-cysteine was also temperature dependent with a 77% reduction in bath-to-cell transport, 76% decrease in cytosolic concentration, and 86% decline in tubular contents at 12°C when compared to 37°C. A significant decrease in transport was seen with the classic organic anion transport (OAT) inhibitors of PAH (71% and 67%) and probenicid (48% and 38%), as well as, the dicarboxylates, adepate (over 100%) and glutarate (69% and 52%) in both bath-to-cell and cytosolic concentration respectively. The addition of L-methionine to the Me203Hg+-L-cysteine conjugate significantly reduced the bath-to-cell transport by 64% and the cytosolic contents by 47%. The Me203Hg+-L-taurine conjugate also demonstrated temperature dependence (99% reduction and 91% decrease in bath-to-cell and cytosolic concentration respectively, at 12°C when compared to 37°C). Inhibition with PAH was also seen (77% reduction) in bath-to-cell transport and 67% decline in cytosolic concentration giving further evidence to the transport of the Me203Hg+-L-taurine conjugate via OAT. When Me203Hg+ was conjugated to L-methionine a 55% reduction in bath-to-cell transport was seen which was also temperature dependent (59% decrease at 12°C when compared to 37°), although no significant decrease in transport was noted with the addition of PAH. Analysis of the methylmercury conjugates via mass spectrometry demonstrated that L-cysteine, L-taurine, and L-methionine all are binding with methylmercury in the same unknown common configuration (MW=409). These results indicate the mercuric conjugate is gaining entry into the renal epithelial cells via the OAT and the amino acid transport system in the basolateral membrane of the proximal tubule.

Choline is indispensable for a number of fundamental processes in the body. Besides being the precursor of the acetylcholine, an important neurotransmitter, choline is found in the cell membrane structure combining with fatty acids, phosphate and glycerol. Its deficiency may result in nervous system disorders, fatty acid build up in the liver, along with increased cholesterol levels, high blood pressure and memory loss. Thus, rapid detection methods are required for the determination of choline in biological fluids. In this study a choline oxidase biosensor was constructed for the determination of choline. During construction of the biosensor, glucose oxidase was used as a model enzyme, before choline oxidase used. The Teflon (PTFE) membrane of the oxygen electrode was grafted with 2-hydroxyethyl methacrylate (HEMA, 15%, v/v) in the presence of ferrous ammonium sulphate (FAS, 0.1%, w/v) by gamma irradiation and ethyleneglycol dimethacrylate (EGDMA, 0.15 %, v/v) was used as a crosslinker in a series of membranes. HEMA-grafted membranes were activated with epichlorohydrin or glutaraldehyde to maintain covalent immobilization of enzyme. The enzyme activity was measured with an oxygen electrode unit based on oxygen consumption upon substrate addition. Membranes were characterized in terms of grafting conditions and mechanical properties. Membranes, gamma irradiated in a solution of HEMA (15%) and FAS (0.1%) for 24 h, were found to be suitable for use in the further studies. Mechanical test results revealed that HEMA grafting made Teflon membrane more flexible and the presence of EGDMA made the grafted membrane stiffer. During optimization stage, it was found that the immobilized enzyme amount was not sufficient to obtain enzyme activity. Thus, the membrane preparation stage was modified to obtain thinner membranes. The immobilized glucose oxidase and choline oxidase contents on thin HEMA grafted membranes were determined by Bradford and Lowry methods. The influence of EGDMA presence and the epichlorohydrin activation duration on enzyme activity studies revealed that the membrane should be prepared in the absence of EGDMA and 30 min activation duration is appropriate for epichlorohydrin coupling. The study on the influence of membrane activation procedures revealed that the membranes activated with glutaraldehyde had a higher specific activity than the membranes activated with epichlorohydrin. Upon stretching membrane on the electrode directly rather than placing in the sample unit, the response of the enzyme immobilized sensor improved with high specific activity. The optimum choline oxidase concentration was found to be 2 mg/mL considering the effect of immobilization concentration on enzyme activity. With the choline oxidase biosensor, the linear working range was determined as 0.052-0.348 mM, with a 40 &plusmn / 5 &micro / M minimum detection limit. The response of the sensor decreased linearly upon successive measurements.

This report details the derivation of an analytical model for a flexible membrane wave barrier. The wave barrier consists of a thin flexible membrane suspended in the water column by a moored cylindrical buoy on the free surface and fixed to a hinge at the seafloor. The analytical model combines the three-degree of freedom rigid body motion of the cylindrical buoy with the two-dimensional analog of a vibrating string for the response of the flexible membrane. Theoretical results for reflection and transmission coefficients, dynamic mooring line tension, horizontal hinge force, horizontal and vertical displacements and rotation of the cylindrical buoy are compared with measured results presented by Bender(1989). In general, the theoretical results compare favorably with measured results for moored systems. However, additional studies are required to more precisely quantify the added mass and radiation damping properties of flexible membranes in oscillating flows. / Graduation date: 1991