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Lipids and Biological Membranes 1. 2. 3. 4. Lipid Classification Lipid Bilayers Membrane Proteins Membrane Structure and Assembly Lipids • Lipos, fat • 4 major group of biol. Molecules: – – – – Nucleic acid Proteins Polysaccharides Lipids • Water insoluble -> Aggregate to form membranes • 3 major functions: – Formation of membranes – Energy storage (fat) – Signaling (intra- and extracellular) Membranes • No life without membranes • Membranes compartmentalize • Lipid/Protein ratio ~1:1 • Lipids are soluble in organic solvents (chloroform, methanol) but not in water – Simple isolation by extraction of tissue with organic solvents – • Lipids are amphipatic: hydrophilic head group and hydrophobic acyl chain tail 1) Lipid Classification A) Fatty Acids B) Triacylglycerol C) Glycerophospholipids D) Sphingolipids E) Steroids F) Other Lipids A) Fatty acids • Carboxylic acids with long hydrocarbon chains, C16, C18, C20,...(even numbered) • Saturated, unsaturated, polyunsaturated – – – – Cis- double bonds Begins at C9, ∆9 Melting temperature ! No conjugation of double bonds • ω-3 and ω-6 PUFAs – Linoleic acid, C18:2, ∆9,12 ‒ α-linoleic acid, C18:3, ∆9,12,15 B) Triacylglycerols adipocyte Triacylglycerols (2) o Oil and fats: neutral lipids o Oils, liquid, plant origin, more unsaturated fatty acids o Fat, solid at room temperature, animal origin, more saturated o Not membrane-forming lipids !! o Energy storage (6x more than glucose): 2-3 months o Intracellular storage in lipid droplets o Adipocytes o Thermal insolation C) Glycerophospholipids o major lipid component of membranes o glycerol-3-phosphate with fatty acids at position 1 and 2, Pi in 3 o Amphiphatic: tail / head o If X = H; phosphatidic acid o sn-1; more saturated fatty acids o sn-2; more unsaturated fatty acids Example: 1-Stearoyl-2-oleoyl-3phosphatidylcholine Lung Surfactant • Mostly dipalmitoyl-PC • Carbohydrate tail into air • Covers extracellular space of alveolar cells • prevents collapse upon expiration • prevents de-hydration Phospholipases hydrolyze glycerolipids o present in bee and snake venom o used to determine the structure of unkown lipids o lipases degrade triacylglycerol o some lipases have signaling function, i.e. PLA2, PLC Model of Phospholipase A2 and a glycerophospholipid • From cobra venome • Ca2+ • Bds surface Plasmalogen o Belong to phospholipids o Have vinyl ether instead of ester linked alkenyl chain o Headgroup: Ethanolamine, choline, serine D) Sphingolipids o Mysterious function = named after Sphinx o No glycerol backbone, but „long-chain-base“: Sphingosine/ Dihydrosphingosin (C18); results from the condesation of serine (C2) with palmitic acid (C16). o Long-chain-base is N-acylated to yield ceramide o Ceramide can carry different headgroups: Sphingomyelin (Myelin membrane !): Ceramide with phophatidylcholine-headgroup, most abundant of the sphingolipids 10-20 mol% of plasma membrane lipids Sphinx o Lion with human head (Pharaonen) o Sphingo, gr. Erwürgen o Wächter/Beschützer zb Tempel stellen Rätsel zum Eintritt Sphingolipids (2) Sphingomyeline Ceramide Complex Sphingolipids • Glycosphingolipids/Cerebrosides: contain single sugar residue; galactocerebroside, glucocerebroside lack phosphate, are non-ionic • Gangliosides, most complex sphingolipids: ceramide with attached oligosaccharide; ganglioside GM1, GM2, GM3 constitute 6% of brain lipids) Gangliosides o Act in cell-cell recognition o Receptor for cholera toxin o Defect in degradation results in Tay-Sachs disease E) Steroids o mostly of eukaryotic origin o tetracyclic structure o Cholesterol: o C3-OH group o 30-40 mol% of plasma membrane lipids (like sphingolipids) o Esterified to cholesteryl ester Cholesterol o weakly amphiphatic due to C3-OH o Plasma membrane lipid Steroid hormones o Cholesterol is precursor to steroid hormones o Glucocorticoids, cortisol, affect carbohydrate, protein, and lipid metabolism, inflammation, made by cortex of adrenal gland o Mineralocorticoids, aldosteron, regulate excretion of salt and water by the kidneys, made by cortex of adrenal gland o Androgens and estrogens affect sexual development and function made by testes and ovaries Vitamin D regulates Ca2+ metabolism o Sterol derived hormone with C9 - C10 bond interrupted o Vitamin D2/D3 (ergo- / choleo-calciferol) is made in the skin through photolytic action of UV light o Further hydroxylated to the active form in the liver (C25) and kidney (C1) o Promotes absorption of dietary Ca2+ for deposition in bones and teeth for mineralization o Deficiency results in rickets children, high doses are toxic F) Other Lipids o Isoprenoids, built from C5 isopren units (terpenoids) precursor to sterols also yields a number of fat-soluble vitamins Ubiquinone (coenzyme Q) electron transport in mitochondria 10x isoprene units Vitamin A (retinol) eye photoreception Derived from β-carotene Other Lipids (2) Eicosanoids are derived from arachidonic acid Derived from C20:4 fatty acid, eicos = 20 act at very low concentrations Act paracrine, on neighboring cells evoke pain, fever, blood pressure released by phospholipase A2 inhibited by aspirin 2) Lipid Bilayers Lipids form aggregates in aqueous solutions: micelles Bilayer formation Bilayer • Shape of lipid monomer determines the type/structure of aggregate formed • Cylindrically shaped lipids form bilayers that can seal to liposomes • Bilayer thickness ~60Å • Liposome diameter ~50nm Liposome B) Lipid mobility • Fast lateral diffusion (ca 1µm/sec) • Slow flip-flop (days) => bilayer as two-dimensional fluid Model of a lipid bilayer Bilayer fluidity is temperature dependent Liquide-crystalline Gel • Phase transition temperature of biological membranes 10-40°C; dependent on fatty acid composition • Tm increases with fatty acid chain length (~15°C/C2 unit) Tm decreases due to desaturation (~50°C/double bond) • Bacteria and und poikilothermic organisms adjust the fatty acid composition to maintain membrane liquid -> homeoviscous adaptation • Cholsterol decreases membrane fluidity Cholesterol in membranes • Cholesterol is by itself not membrane forming • Reduces fluidity by reducing mobility of fatty acids • Prevents formation of gel phase by preventing aggregation of fatty acids • Broadens Tm 3) Membrane Proteins • Biological membranes: Lipid/Proteins ~1:1 • Proteins provide functionality, ion transport • Proteins required for biogenesis of membrane; no de novo formation of biological membranes A) Integral membrane proteins Classes of membrane proteins: – Integral/intrinsic; thigthly associated with membrane, can only be removed by detergents (SDS) – Peripheral membrane proteins; can be dissociate from teh membrane by high salt (1.5M NaCl) Glycophorin A Integral membrane proteins are amphiphiles and asymmetrical: Glycophorin A has 3 domains: External, 72aa, 16 carbohydrates Transmembrane (TMD), 19aa, hydrophobic aa, spans membrane Cytosolisch, 40aa, charged and polar aa Hydrophobicity-blot of Glycophorin A Bacteriorhodopsin Model of a complex, „polytopic“ membrane protein – hallobakterium halobium (Purplemembrane) – Death Sea, 4.3M NaCl – 247aa, light-driven proton pump generates membrane potential to drive ATP synthesis – Retinal is light-absorbing group (similar to Rhodopsin in our eye) Bacteriorhodopsin (3) Richard Henderson & Nigel Unwin o used electron crystallography to solve the structure of bactreriorhodopsin ≠ X-ray o Purple membrane: 2 dimensional crystall of bactreriorhodopsin 75% with 25% lipids o Small (248aa) stable protein o First report in 1975, 3.5Å/10Å resolution Electron crystallography • Structural analysis of solid surfaces (regular patterns by electron microscopy • ≠ X-ray crystallography – 7 helices, each ~25aa Electron Crystallography Bacteriorhodopsin (2) Porin • outer membrane of Gram-negative bacteria • outer mitochondrial membrane • trimer, 30-50 kD units • 16- stranded antiparallel beta sheets βbarrel (Fass) • pore diameter 7Å, length ~55Å B) Lipid-Linked Proteins • Some membrane proteins contain covalently attached lipids that anchor them to the periphery of the membrane. • 3 classes: – Prenylated proteins (farnesyl C15, and geranylgeranyl, C20) – Fatty acids (Myristoyl, Palmitoyl) – Glycosylphosphatidylinositol (GPI) • reversibel/irreversibel membrane-anchor, targeting to outer or inner leaflet of plasma membrane Prenylated Proteins • C-terminal prenylation site: CaaX box, prenyl is thioether-bound to Cys • Isopren (C5) is monomeric unit for farnesyl- (C15) and geranylgeranyl- (C20) anchor Acylated Proteins • myristoylated (C14), co-translational, Nterminal Gly, amide-bond, stable/ irreversible • palmitoylated (C16), post-translational, thioester bond to Cys, reversible, targeting to cytosolic surface of plasma membrane, frequently combined with myristoylation or prenylation GPI-anchored Proteins • Anchoring of proteins to the outer leaflet of the plasma membrane, Surface of cells, cleavable. • C-terminal to proteins, anchoring in the lumen of the ER. C) Peripheral Membrane Proteins • Can be released from the membrane by salt or pH changes • Bind to membranes by non-covalent interactions with lipid-headgroups, i.e. cytochrome c 4) Membrane Structure and Assembly • How do proteins and lipids assemble to form a biological membrane ? • Singer and Nicholson, 1972: The fluid mosaic model • Proteins float in a 2-dimensional sea of lipids, similar to „icebergs“ in the sea => Free lateral diffusion of proteins Experiments: Cell fusion FRAP Model of the Plasma Membrane Cell fusion experiment FRAP Fluorescence recovery after photobleaching B) The Membrane Skeleton • Erythrocyte as model, easy to isolate, no organelles • Membranous bag of hemoglobin • Osmotic lysis -> ghost • Bikonkave Form • But membrane flows over an underlying cytoskeleton • Spectrin, 75% of cytoskeleton α (280 kD), β (246 kD) subunits 106aa repetitive unit form triple-stranded helical coil-coil αβ dimer is up to 1000Å long head to head assoc. to (αβ)2 tetramer to form protein meshwork below the membrane, interacts with ankyrin Muscular dystrophy / Hereditary spherocystosis Spectrin Mobility of Membrane Proteins Gates and fences model C) Lipid Asymmetry o Lipids and proteins are asymmetrically distributed across the bilayer o Carbohydrate portion of proteins and glycolipids face exterior of cell o PS is in cytosolic leaflet o Experimental determination o Phospholipase D treatment o Pulse labelling + chemical modification Lipid Asymmetry (2) Model of the Plasma Membrane Sphingomyelin PC PS PE Lipid Synthesis o Lipids are made by integral membrane enzymes - in the ER of eukaryotic cells (smooth ER) - at the plasma membrane of prokaryotes o how does a membrane expands upon lipid synthesis ? (symmetrically or asymmetrically ?) => Rothman and Kennedy experiment: Lipid Asymmetry (3) o TNBS to chemically modify PE Lipid Asymmetry (4) o The topology of lipid synthesis (in bacteria): Lipid Asymmetry (5) o fast equilibration of newly made lipids between the two leaflets of the bilayer (3 min) o but slow rate of lipid flip-flop in protein free membranes => equilibration is catalyzed by phospholipid “flipase” (facilitated diffusion) By phospholipid “translocase”; requires ATP, translocation against a gradient (active transport) Membranes are generated by expansion of existing membranes, no de novo formation ! Lipid Rafts o are membrane subdomains, rich in sterols and sphingolipids o lateral heterogeneity within membrane, platforms o act in: signal transduction (clostering of receptors) protein/membrane sorting o epithelial cells are polarized: apical / basolateral surface, which have different lipid and protein composition D) The secretory pathway o How are membrane proteins synthesized ? o 25% of all proteins are integral membrane proteins o How do these proteins reach the cell surface o In eukaryotes / prokaryotes ? o How are extracellular proteins secreted ? => Proteins are synthesized in the rough ER (RER) and are then transported by membranous carriers, vesicles, to the cell surface The secretory pathway (2) o Unlike lipids, membrane proteins cannot flip across the membrane o Proteins are synthesized from an mRNA template that is read by the ribosome to condense/polymerize amino acids - N->C terminal growth of protein o Free Ribosomes synthesize soluble proteins o Membrane proteins are made by ribosomes at the rough ER (RER) (about 40% of all proteins are made at the ER membrane (integral membrane & secreted proteins) The secretory pathway (3) 1) All secreted, ER-resident, lysosomal proteins, and many TM proteins contain a signal peptide at the N-term 13-36 Aa, hydrophobic 2) This signal peptide is recognized by the signal recognition particle, while the protein is being made by the ribosome, translational arrest 3) The arrested ribosome is targeted to the signal recognition particle receptor (SRP receptor) 4) Translation continues trough a channel in the ER membrane, the translocon N-Terminal sequences of some eukaryotic secretory pre-proteins How does the ribosome know whether it makes a soluble or membrane protein ? The signal sequence Co-translational Translocation The docked ribosome Sec61/SecY: the translocon Release of TMDs by the translocon E) Intracellular vesicles transport proteins Requires: 1. Packaging of cargo proteins into membrane vesicles, vesicle coat assembly, vesicle budding 2. Fusion of the vesicle with a defined target membrane, controlled by SNARE proteins E) Intracellular vesicles transport proteins Shortly after their synthesis at the RER, secreted proteins reach the Golgi (cis, medial, trans) and then the plasma membrane In the Golgi, proteins are step wise modified, i.e. through glycosylation Vesicular transport Membrane, secretory, and lysosomal proteins are transported in Coated Vesicle Membranous carriers: vesicles Coated by proteins: COPII, COPI (coat proteins) Clathrin (polyhedral) 60-150nm diameter Anterograde (ER -> Golgi) Retrograde (Golgi -> ER) Vesicle formation by protein coating COPII, ER->Golgi Sec24,24 and Sec13,31 COPI, intra Golgi, Golgi->ER Heptamer Clathrin, Golgi -> PM/Endosome PM -> Golgi/Endosome Lelio Orci, Geneva Vesikel fusion preserves topology The cell exterior is topologically equivalent with the Golgi lumen and with the ER lumen Clathrin Membran Coat: Protein Mantel um die Membran, um sie abzuschnüren Triskelion Polyhedral cage with 12 faces Endocytosis o Exocytosis: transport from ER via Golgi to plasma membrane o Endocytosis: uptake from plasma membrane transport to Golgi and/or lysosome o Fluid phase / receptor / membrane internalization from the cell surface o Clathrin-mediated F) Proteins mediate vesicle fusion o In all cells: New membranes are generated by the expansion of existing membranes o In eukaryotes: through vesicular transport of proteins & lipids o Vesicles are formed by coat proteins o How do vesicles fuse with target membrane ? Vesicle fusion o Studied in yeast and in neurons (synapse, neurotransmitter release) o Biological membranes do not spontaneously fuse o Requires SNARE proteins SNAREs o confer fusion specificity (?) o catalyze fusion (?) SNAREs form a 4-helix bundle o SNAREs mediate vesicle fusion o Cleaved by tetanus or botulinus toxins Tetanus and botulinum toxins specifically cleave SNAREs o Tetanus, wound contaminations; Botulism, food poisoning o Made by anaerobic bacteria of genus Clostridium o 10 Mio times more potent than cyanide o TeTx, BoNT/A – BoNT/G (serotypes): uptake by specific neurons through endocytosis o SNARE cleavage, halts exocytosis, paralysis o “Botox”, relieves chronic muscle spasms, cosmetics (3 months) Viral fusion proteins o How do viruses enter cells ? Example: Influenza Virus 1. Host cell recognition (specificity) 2. Activation of viral membrane fusion machinery 3. Fusion of virus with host cell membrane, release viral genome into host cytosol o Major integral membrane protein of virus envelope: Hemagglutinin (HA) (agglutinates erythrocytes) After binding to cell surface -> endocytosis, endosomes, pH drop (~5) => triggers conformational change on HA to expose a “fusion peptide” Influenza Virus pH-dependent conformational change on HA triggers membrane fusion Influenza Virus