1,290 research outputs found
Solubilization of lipids and membrane proteins into nanodiscs: Mode of action and applications of SMA copolymers
No full textCell membranes separate the inside and outside of cells. Membrane proteins in the cell membrane control the traffic of molecules across the membrane and are therefore targets for a lot of drugs: about 50 % of all approved drugs target a membrane protein! Unfortunately, scientists only know little about membrane proteins as compared to, for example, water soluble proteins. That is because membrane proteins are hard to study since they reside in the hydrophobic patch of the membrane. To study membrane proteins, they need to be isolated and detergents (soap) are required to do so. The problem is: soap molecules wash away and replace the native membrane environment. In that way, many membrane proteins are destabilized by detergents. In this thesis, a novel method is described that overcomes the issues that detergents bring to the study of lipid membranes and membrane proteins. And help comes unexpectedly. It is the synthetic industrial polymer called styrene-maleic acid, which is often used in the car industry, that is able to isolate membrane protein in a very soft manner. Upon the addition of SMA to membranes, the membranes solubilize in nanodisc particles in which a membrane protein is captured along with a small piece of its native lipid environment. This thesis uses a photosynthetic protein named ‘reaction center’ (RC) form the purple bacterium Rhodobacter sphaeroides as model to validate SMA polymers as alternative to soap molecules. The results show that the RC in SMA nanodiscs maintains the native lipid environment, which is in contrast to soaps such as LDAO and DDM. Furthermore, RCs in a nanodisc are more heat and light stable than RCs in soap and in the native membrane. That opens options to use RCs in SMA nanodiscs in bio-solar cells for which protein stability is key. More results give insight in the molecular action of membrane solubilization by SMA polymers. For example, SMA is a very efficient solubilizing agent, much more than for example membrane scaffold proteins (MSPS, which is the conventional way to produce nanodiscs. Also, the SMA variant with a 2:1 styrene-to-maleic acid ratio is the best SMA variant to use. That is because this polymer has the optimal balance in hydrophobic and hydrophilic groups to attack and solubilize lipid membranes, while stabilizing nanodiscs in water. SMA was also found to solubilize the exact membrane lipid composition in nanodiscs, i.e. SMA has no preference to solubilize certain lipid species. This is a very important result, because thanks to this, SMA is the first way to study lipid-lipid and lipid-protein interactions in a direct and biochemical way. The SMA technique is still young (since 2009 reported in the scientific literature) and many concepts need still to be explored. But due to the potential of SMA to solubilize and stabilize membrane proteins, it is possible that purification of membrane proteins using SMA could become the standard tool for biophysical studies of membrane proteins, and that the polymer will greatly facilitate the wider use of SMA nanodiscs in biohybrid devices
Lipidomics in research on yeast membrane lipid homeostasis
Get PDFMass spectrometry is increasingly used in research on membrane lipid homeostasis, both in analyses of the steady state lipidome at the level of molecular lipid species, and in pulse-chase approaches employing stable isotope-labeled lipid precursors addressing the dynamics of lipid metabolism. Here my experience with, and view on mass spectrometry-based lipid analysis is presented, with emphasis on aspects of quantification of membrane lipid composition of the yeast Saccharomyces cerevisiae. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein
The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets
No full textPeptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs), have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated) proteins in bacterial cells and the perspectives on how to overcome the issues
Insights into membrane solubilization by styrene-maleic acid copolymers
No full textStyrene-and-maleic acid copolymers (SMA) are gaining interest in membrane protein research due to their ability of solubilizing lipid membranes into nanodiscs. Using commercially available SMA preparations it was found that SMA blends are fully promiscuous when mixed with membranes and that any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside. Furthermore, we demonstrate that the average length and hydrophobicity are important parameters in determining solubilization efficiency and properties of the resulting nanodiscs. Low Mn polymers were found to insert to a higher extent into lipid monolayers and solubilized most efficiently lipid vesicles. On the other hand, nanodiscs bounded by high Mn polymers were found to be more stable, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates between lipids in nanodiscs. Stability was further improved by using polymer blends with a relatively low styrene content. Finally, it was found that SMA-bounded nanodiscs “breathe”, allowing expansion of the enclosed lipids. Hence, these nanodiscs most likely allow conformational changes of membrane proteins embedded in them. Overall, this thesis contains systematic studies that contribute to understanding, optimization and further development of a new procedure to reconstitute membrane proteins into nanodiscs: the SMA solubilization approach
Towards novel antibacterial development: from peptidoglycan to lipoprotein biogenesis
No full textAntibiotic resistance has been an emerging threat to the public health. Since the discovery of penicillin reported in 1929, continuous effort has been made in the past decades to discover more effective antibacterial molecules through drug-target interactions and molecule modifications . Antibiotic-mediated cell death starts with physical interaction between the drug molecule and its specific target that, depending on its mode of action, induces DNA damage, protein misfolding and mistranslation, cell envelope damage, and loss of structural integrity of the membrane(s). In addition, it was reported that major classes of bactericidal antibiotics induce cell death through the production of highly toxic hydroxyl radicals in both Gram-negative and Gram-positive bacteria, regardless of drug-target interaction. These results point out that the mechanism by which the current antibiotics kill bacteria is multilayered and complex. Bacterial cell envelope provides a rich source of drug targets and at the mean time also a barrier for certain antibacterial treatment. The major part of this PhD thesis describes the characterization of the phospho-MurNAc-pentapeptide translocase MraY, an enzyme that catalyzes the first membrane step of peptidoglycan synthesis. Being an alpha-helical membrane protein, MraY had been challenging to produce and purify with high yield. Characterization of MraY was also hampered due to lack of a crystal structure until very recently. Kinetics studies reported in the literature presented controversial evidences what the catalytic mechanism is. Furthermore, the development of MraY inhibitor was not very successful because the enzyme has two substrates, one being membrane embedded and not well documented, while the other resides in the cytoplasm, which is difficult to reach. In this PhD thesis, thorough kinetics experiments by varying the concentration of both substrates were conducted. The true kinetics values of this two-substrates enzyme were obtained. It is found that MraY must bind concomitantly to both substrates before the release of either of its products. Docking experiments gave insights to the binding model of MraY to its lipid substrate. The role of a catalytically important histidine residue was given. Furthermore, by using a novel styrene maleic acid copolymer system instead of the conventional detergent system to produce and characterize MraY, it was demonstrated that the accessibility of the embedded MraY to the lipid substrate can largely influence the starting rate of the MraY-catalyzed reaction. In the second part of the PhD thesis, a split-SNAP fluorescent reporter system for the localization of E. coli outer membrane lipoprotein is described. It was demonstrated that it is possible to detect defects along the lipoprotein biogenesis pathway using such a system. By modifying the constructs, the exploitation of such a reporter system can be expanded to outer membrane protein biogenesis as well. Finally in the last part, the overview of a promising alternative approach, antibacterial photodynamic therapy, to combat resistant bacterial strains was given. In this context, the bacterial cell envelope not only provides targets but also barriers for the delivery of the photosensitizers
Phosphatidylcholine's functions beyond that of a membrane brick
Get PDFSince its discovery in the 19th century, phosphatidylcholine (PC) has been regarded primarily as a structural lipid. However, more recent evidence, much of it in the last five years, strongly suggests that PC has other roles. Here, we explore some of that new evidence and consider the possibility that the ultimate role of phosphatidylcholine may not be predictable
The styrene-maleic acid copolymer:: a versatile tool in membrane research
Get PDFA new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene-maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein-lipid and protein-protein interactions
Towards the structural characterization of proteins involved in peptidoglycan biosynthesis
No full textThe cell wall is an essential structure for bacterial survival and unique to bacteria. It is responsible for maintenance of cellular shape and allows the bacterium to withstand high differences in osmotic pressure between the inner and outer leaflet of the cell. Consequently, the bacterial cell wall has been an optimal target for the development of antibiotics for over half a century, particularly the assembly of its major structural component, the peptidoglycan. Despite the progress in understanding the structure and function of several enzymes involved in the biosynthesis of peptidoglycan, however, the mechanism and regulation of their intermolecular assembly is still not fully understood. In this thesis, we use several biochemical and structural approaches to gain more insight into the complex process of peptidoglycan biosynthesis and its regulation. The first part of this thesis describes the characterization of an interaction between proteins that exert antagonistic functions during cell wall growth. By surface plasmon resonance we show that Pseudomonas aeruginosa PBP2 and SltB1 interact in a highly Ca2+-dependent manner. Structural determination of SltB1 revealed an EF-hand motif that might be required for the interaction of the two proteins. The second part of this thesis not only presents strategies to express and purify multi-spanning membrane proteins of the SEDS protein family, but also provides the first biochemical evidence that Streptococcus pneumoniae RodA functions as Lipid II flippase, a role which has to date only been reported for Escherichia coli FtsW. In addition, we crystallized RodA and obtained initial diffraction data. Determination of its three-dimensional structure will not only provide insight into the mechanism of lipid transport across cellular compartments but will also open up new ways for rational drug design
The role of phospholipid molecular species in determining the physical properties of yeast membranes
No full textIn most eukaryotes, including Saccharomyces cerevisiae, glycerophospholipids are the main membrane lipid constituents. Besides serving as general membrane 'building blocks', glycerophospholipids play an important role in determining the physical properties of the membrane, which are crucial for proper membrane function. To ensure optimal physical properties, membrane glycerophospholipid composition and synthesis are tightly regulated. This review will summarize our current knowledge of factors and processes determining the membrane glycerophospholipid composition of the reference eukaryote S. cerevisiae at the level of molecular species. Extrapolating from relevant model membrane data, we also discuss how modulation of the molecular species composition can regulate membrane physical properties
Furan fatty acids efficiently rescue brain cells from cell death induced by oxidative stress
No full textTreatment of rat brain C6 astroglioma cells with furan fatty acid F6 prior to exposure to hydrogen peroxide shows a strong protective effect of F6 against cell death resulting from oxidative stress. This protective effect is obtained only for F6 administered as a free fatty acid and with an intact furan ring. It is proposed that brain cells are rescued by F6 scavenging radicals elicited by lipid peroxidation within the cell membrane. Oxidative processes outside the cell membrane, such as protein carbonylation, are not affected by F6. Furan fatty acids such as those present in fish oils and marine organisms are likely beneficial for consumption in reducing the risk of diseases that have been implicated to arise from oxidative stress, such as Alzheimer's disease
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