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Lecture 9 MEMBRANES Structure & Dynamics [email protected] http://glutxi.umassmed.edu/ S1-824 (a.m.), LRB 926 (p.m.) 6-5570 The Cell Membrane http://www.youtube.com/watch?v=Qqsf_UJcfBc Objective of Class • To emphasize that biomembranes comprise noncovalent, self-assembling, multicomponent, dynamic, macromolecular assemblies of amphipathic molecules. Specific Goals This lecture reviews: • Biomembranes • Lipid Structure • Physical Properties of Lipids • Physical properties of lipid bilayers • Properties & uses of detergents • Lipid distribution within and across bilayer leaflets (From Bloom and Fawcett, A Textbook of Histology, Chapman and Hall, N.Y., 12th edition, 1994, Figure 1-2.) Membranes separate the cell from the outside world and separate organelles inside the cell to compartmentalize important processes and activities. Cellular membranes have diverse, location-specific functions within the cell. At the electron microscopic level, membranes share a common structure following routine preparative steps. The figure above shows a typical "Unit" membrane which resembles a railroad track with two dense lines separated by a clear space. This figure was obtained by cell fixation/sectioning and staining with osmium tetroxide (an electron opaque agent that binds to a variety of organic compounds). This figure actually shows two adjacent plasma membranes, both of which have the "unit membrane" structure. Membranes can be isolated in several ways. • Red cells are simply a plasma membrane plus cytosolic proteins - hypotonic lysis of red cells produces cytoplasm-free membranes. • Other, more complex cells must be homogenized and membrane fractions isolated by density centrifugation (organelle membranes and fragments have densities distinct from those of plasma membranes). Analysis of the composition of isolated membranes indicates that the major constituents are: 1. lipids (phospholipids and cholesterol) 2. proteins 3. carbohydrates. Lipid Structure H2C H C glycerol CH2 OH OH OH OH O P H O H2C OH OH CH2 OH glycerol-3-phosphate Hundreds of different kinds of fatty acids are found. Some have 1 or more double bonds in their hydrocarbon tail and are called unsaturated. Fatty acids with no double bonds are saturated. This double bond is rigid and creates a kink in the chain. The rest of the chain is free to rotate about the other C-C bonds. In nature most unsaturated fatty acids are cis fatty acids, meaning the hydrogen atoms are on the same side of the double carbon bond. In trans fatty acids the two hydrogen atoms are on opposite sides of the double bond. Table 11-1 The Common Biological Fatty Acids Symbol" Common Name Saturated fatty acids 12:0 Lauric acid 14:0 Myristic acid Palmitic acid 16:0 18:0 Stearic acid Arachidic acid 20:0 22:0 Behenic acid 24:0 Lignoceric acid Systematic Name Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Octadecanoic acid Eicosanoic acid Docosanoic acid Tetracosanoic acid Unsaturated fatty acids (all double bonds are cis) 16:1 Palmitoleic acid 9-Hexadecenoic acid 18:1 Oleic acid 9-0ctadecenoic acid 9,12 -Octadecadienoic acid Linoleic acid 18:2 9,12,15-0ctadecatrienoic Q- Linolenic acid 18:3 acid 6,9,12 -Octadecatrienoic y-Linolenic acid 18:3 acid 5,8,11,14-Eicosatetraenoic Arachidonic 20:4 acid acid 5,8,11,14,1720:5 EPA Eicosapentaenoic acid 15 -Tetracosenoic acid 24:1 Nervonic acid Structure CH3(CH2)lOCOOH CH3(CH2)12COOH CH3(CH2)14COOH CH3(CH2)16COOH CH3(CH2)lSCOOH CH3(CH 2h oCOOH CH3(CH2h2COOH CH3(CH2hCH= CH(CH 2hCOOH CH3(CH2hCH=CH(CH2)7COOH CH3(CH2MCH=CHCH2MCH2)6COOH CH3CH2(CH =CHCH2h(CH2)6COOH mp ("C) 44.2 52 63.1 69.6 75.4 81 84.2 -0.5 13.4 -9 -17 -49.5 -54 39 • Number of carbon atoms: Number of double bonds. Source: Dawson, R. M. Clarendon Press (1969). c., Elliott, D. c., Elliott, W. H., and Jones, K. M., Data for Biochemical Research (2nd ed.), Chapter 11, R2 O O H2C O O R1 a glycerophospholipid CH2 H O O P X O- The common Classes of Glycerophospholipids O O H2CH CO O C C R1R 2 1 O O R2 C CO O CHCH R2 OO H2CH2C O OP P OO RR 33 O-ONAME OF R3 Water Ethanolamine Choline Serine FORMULA OF R3 NAME OF PHOSPHOLIPID –H –CH2CH2NH3+ Phosphatidic acid Phosphatidylethanolamine –CH2CH2N(CH3)3+ Phosphatidylcholine (lecithin) –CH2CH(NH3+)COO- Phosphatidylserine H OH myo-inositol H Glycerol OH H OH OH H H OH Phosphatidylinositol H Phosphatidylglycerol –CH2CH(OH)CH2OH O Phosphatidylglycerol –CH2CH(OH)CH3 O P O CH2 R4 OHC O C O CH2 Diphosphatidylglycerol (cardiolipin) O O C R5 CH3 H3C N+ CH3 CH2 CH2 O -O P O O H H2C C CH2 O O C C ester linkage O O (CH 2)7 (CH2)14 CH CH3 CH (CH2)7 CH3 1-Palmitoyl-2-oleoyl-3-phosphatidylcholine In nature most unsaturated fatty acids are cis fatty acids, meaning the hydrogen atoms are on the same side of the double carbon bond. In trans fatty acids the two hydrogen atoms are on opposite sides of the double bond. CH3 H3C N+ CH3 CH2 CH2 O O H O- O H2C O P CH2 O O C C O (CH2)16 (CH2)16 CH3 CH3 1,2-Distearoyl-3- phosphatidylcholine Text Sphingosine H3C (H2C)12 C H C H H2 C H C H C OH NH3+ OH Sphingomylein O H3C (H2C)12 C H H3C C H H C H C OH NH (H2C)14 C H2 C O P O H2 C H2 C N+ CH3 CH3 phosphoryl choline unit CH3 O- amide linkage O fatty acid unit Cerebroside (a glycolipid) H OH H3C (H2C)12 C H H3C C H H C H C OH NH (H2C)14 C H2 C H O O H O H OH fatty acid unit H OH OH glucose or galactose unit CH3 HC CH3 CH3 HO Cholesterol CH3 C H2 C H2 C CH H2 CH3 TABLE 10–1 Approximate Lipid Compositions of Different Cell Membranes PERCENTAGE OF TOTAL LIPID BY WEIGHT LIPID LIVER CELL PLASMA MEMBRANE Cholesterol Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Sphingomyelin Glycolipids Others 17 7 4 24 19 7 22 RED BLOOD CELL PLASMA MEMBRANE 23 18 7 17 18 3 13 MYELIN 22 15 9 10 8 28 8 MITOCHONDRION (INNER AND OUTER MEMBRANES) 3 25 2 39 0 trace 21 ENDOPLASMIC RETICULUM 6 17 5 40 5 trace 27 Note, molecular weight of cholesterol = 386.7 while that of a typical PC ≈ 760. Thus a phospholipid : cholesterol ratio of 1 : 1 by mass ≈ 1 : 2 as a molar ratio. E. COLI BACTERIUM 0 70 trace 0 0 0 30 Physical properties of lipids & lipid bilayers Early cell biologists deduced membrane structure from electron microscopic images and the knowledge that membranes were lipoprotein complexes. They surmised that the electron-opaque material included phospholipid headgroups and proteins and that the electron-transparent membrane material comprised phospholipid acyl chains. lipid packing is governed by hydrophobic interactions At the air-water interface, the hydrophobic tails of a lipid monlayer avoid association with the water by extending into the air. Franklin’s Experiment 1 cruet = 1 cubic m = area = volume on lake = area = thickness = volume/area 2 mL 1000000 mL 0.5 acres 2023 m2 2 mL 2 x 10-6 m3 2023 m2 9.89E-10 m Franklin investigated the way in which oil could be used to calm water surfaces. He first performed this experiment on Clapham Pond in the summer of 1771, and subsequently carried a cane containing a small oil holder to repeat his "conjuring trick" on his travels. He stopped short of computing the thickness of the mono-layer. see: http://www.rsc.org/learn-chemistry/content/filerepository/CMP/00/000/687/ isms-9.pdf?v=1399160447243 lipid packing is also governed by lipid shape Molecular Molecular shape packing lipid micelle water lipid bilayer The cylindrical shape of phospholipids causes them to form extended, disk-like micelles that are best described as lipid bilayers. Lysolipids and detergents containing only a single acyl chain form micelles. Modified from Molecular Biology of the Cell, 4th edition Energetically unfavorable phospholipid bilayer edges exposed to water sealed phospholipid vesicle no edges exposed to water Energetically favorable Modified from Molecular Biology of the Cell, 4th edition A. Electron micrograph of a multilamellar phospholipid vesicle in which each layer is a lipid bilayer (After Bangham, Cambridge Univ) B. An electron micrograph of a liposome. Its wall, as the diagram indicates, consists of a bilayer (After Stoekenius, UCSF) Question How do we know that the lipid component of biological membranes is assembled into lipid bilayers? Supporting evidence # 1 The staining pattern of plasma membranes obtained using osmium tetroxide persuaded scientists that the unstained inner core of the membrane lacked proteins. Hence, it was assumed that membrane proteins formed beta strands that coated the lipid bilayer. In 1966, Lenard and Singer, using CD, observed that > 30% of membrane proteins are α-helical. This made it likely that there were many spherical proteins not just beta strands. Singer studied phospholipid bilayers and found that they form a flattened surface on water, with no requirement for a protein coat. The turning point in the modeling came with the advent of freeze fracture techniques. This method shows the inside of a membranes and their "bumps, grooves, ridges". These were later found to be proteins. Supporting Evidence # 2 Paraffin Waxes consist mostly of straight chain hydrocarbons and are available in a wide variety of melting points ranging from 120 to 160 degrees fahrenheit. Paraffin waxes are mainly identified in the candle industry by melting point and oil content. Wax melting is an endothermic reaction (heat is absorbed from surroundings). The 3 types of endothermic phase changes are the transition from solid to liquid, the transition from liquid to gas, and the transition from gas to plasma. The reverse phase changes are exothermic (heat is released to the surroundings). Monitoring the melting process by Differential Scanning Calorimetry Inert reference Actual sample DSC measures the energy needed to establish a nearly zero temperature difference between a substance and an inert reference material. Sample and reference are subjected to identical temperature regimes and are heated or cooled at a controlled rate. Heat flow Heat flow Temp sensor Feedback circuitry to ensure ∆T = 0 Temp sensor The temperatures of the sample and reference are monitored and controlled independently using separate temperature sensors and heat sources. The temperatures of the sample and reference are made identical by varying the power output from the heat sources. The energy required to do this is a measure of the enthalpy or heat capacity changes in the sample relative to the reference. inert sample material freezing a wax (scan down in T) endothermic exothermic Heat flow melting a wax (scan from low to high temp) Temperature In fact the baselines of the meting and freezing curves would be superimposable - they are separated here for illustration purposes only. Sketch of a lipid bilayer, corresponding electron density profile (−) and definitions of structural parameters. Due to the soft-matter character of the bilayer stack, features such as headgroup peaks and methyl trough region are smeared out. In a coarse description using stepwise constant electron densities (−), one can distinguish water, lipid headgroup, hydrocarbon and methyl trough regions 30 20 Dipalmitoyl- phosphatidylcholine bilayers 40 DSC 50˚C endothermic 1250 Molecular Volume Å3 1200 note that the increase in molecular volume ≈ 110 Å3 /molecule ≈ 0.1 m3/ mol ≈ 66 cm3/mol 1150 we will return to this later 1100 P L L X-RAY 67 64 60 carbon chain packing 20 30 40 Temperature (˚C) 50˚C endothermic lipids Heat flow (isolated from membranes & formed into artificial bilayers) proteinase K-treated membranes (note: these records are displaced in the y-axis to aid comparison) exothermic first scan second scan 10 20 30 40 50 60 Temperature, ˚C Mycoplasma laidlawii grown on palmitate From Melchior et al., (1970) BBA 219, 114-122 Supporting Evidence # 3 Low-angle x-ray diffraction analysis of myelin membranes This technique measures the density of matter and can be used to determine the distribution of lipid and protein in biomembranes. (a) During development of the nervous system, a large Schwann cell envelops the axon of a neuron. The continuous growth of the Schwann cell membrane into its own cytoplasm, together with rotation of the nerve axon, results in a laminated spiral of double plasma membranes around the axon. Mature myelin, a stack of plasma membranes of the Schwann cell, is relatively rich in phospholipids. (b) The profile of electron density — and thus of matter — obtained by x-ray diffraction studies on fresh nerve, and the relation of this profile to the protein and lipid components of the myelin membranes. [Adapted from W. T. Norton, 1981, in G. J. Siegel et al., eds., Basic Neurochemistry,3d ed., Little, Brown, p. 68.] Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000. Lipid bilayers are dynamic noncovalent structures lipids diffuse ≈ 1 µm / sec 1 µm = 1 x 10-4 cm k ≈ 2 Dm/λ2 t0.5 = 0.693/k for λ = 3.5 µm k = 0.164 s-1 and t0.5 = 4.23 sec τ = 6 sec Lipid immiscibility A series of fluorescence micrographs of vesicles and monolayers of a 1:1 molar mixture of Dipalmitoylphosphatidylcholine (C16) and dioleoylphosphatidylcholine (C18:1) with varying [cholesterol] in molar %. The fluorophore (Texas Red Dipalmitoyl Phosphatidyl Ethanolamine) is concentrated in cholesterol-poor domains From Veatch & Keller, Phys RevLett 89:268101. 36 detergents - a primer Surfactants are compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents and dispersants. 37 physical properties of detergents Sodium dodecyl sulfate is a detergent with a charged hydrophilic sulfate head group and a 12 carbon hydrocarbon tail. Upon dissolving in water at room temperature (298ºK) it assembles spontaneously (ΔGº = -16.4 kJ/mol) into a higher ordered micelle with the structure shown below OO -O S O S S O O O O O -O O O -O S SDS -O O O S O O O O O O S -O -O O- O O O S O O S polar O S O O O O -O S O O S O O -O -O S O S O O -O S ΔGº = -16.4 kJ/mol = -3.917 kcal/mol O at 20ºC, Keq = 10-∆Gº/1.36 = 759 O O O O O S O S O O - O- O O O O S O O- O- The critical micelle concentration (CMC) is defined as the concentration of surfactant above which micelles form and all additional surfactants added to the system form micelles -O O non- polar O O O 38 HLB is the hydrophilic/lipophilic balance of a molecule and is calculated as: HLB = 20 * MH/M here MH is the molecular mass of the hydrophilic portion of the molecule, and M is the molecular mass of the whole molecule, giving a result on a scale of 0 to 20. 39 An HLB value of 0 corresponds to a completely lipophilic/hydrophobic molecule, and a value of 20 corresponds to a completely hydrophilic molecule. 40 detergent aggregation number Above the cloud point temp (CMT), nonionic detergents become cloudy and phase separate into a detergent-rich layer and an aqueous layer. The temperature at which this occurs is called the cloud point. 41 Questions regarding detergents • What do you think the effects of a detergent on a membrane would be? • What might the advantages of a high CMC? • How do you think a large aggregation number might present a disadvantage? • How do you think we could use CMT to our advantage? Effects of detergents on membranes • Detergents solubilize lipid bilayers and integral membrane proteins. This makes detergents v useful in the purification of membrane proteins. • Solubilization involves several intermediate states that have been studied by a variety of physicochemical and kinetic methods. • • • • Solubilization begins by destabilization of the lipid component of the membranes • In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. In general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. Detergents insert into the membrane (mass action and hydrophobicity/partitioning). Detergent insertion transitions from a noncooperative to a cooperative process. This leads to the formation of membrane fragments of proteins and lipids with detergentshielded edges. + + + Biochim Biophys Acta. 2000 Nov 23;1508(1-2):86-111. + Interaction of membrane proteins and lipids with solubilizing detergents. le Maire M, Champeil P, Moller JV. Explaining the basis of the hydrophobic effect -43 The partition coefficient Nobel, 1974 shows that chemical potential of j (µj) µj = µjo + R T lnCj + Zj e F ψ + V j P + mj g h µjo = chemical potential of substance j in standard state when ψ = 0, h = 0, P and T are standard and Cj = 1M in a particular solvent. As gravity and ∆P unimportant here, µj = µjo + R T lnCj + Zj F e ψ 44 Imagine glycerol is added to a mixture of oil and water. The mixture is shaken until the concentrations of glycerol in oil and water no longer change (equilibrium is achieved). The mixture is allowed to stand (phase separation occurs) and the oil and water phases are assayed for glycerol content. At equilibrium, glyceroloil is in equilibrium with glycerolwater i.e. µjoil = µjwater As glycerol is uncharged, an electrical term is not needed and µ oj oil + RT ln C j oil = µ oj water + RT ln C j water µ oj oil − µ oj water = RT (lnC j water − lnC j oil) or K oil/water = exp[( µ oj water − µ oj oil) / RT ] i.e. K is determined by differences in standard state chemical potential of j in oil and water 45 Koil/water = exp [(µjowater - µjooil)/RT] each µjo determined by energetics of interaction between j and solvent glycerol has three - OH groups resulting in strong Hbonding to H2O and is thus in a more energetically favorable state in H2O ∴ µjowater < µjooil ∴ Koil/water < 1. 46 Advantages of a high CMC • Dilution of [detergent] to values below the cmc results in the dissociation of micelles to individual detergent monomers. • Detergent monomers are much smaller than detergent micelles, and as a result can be easily removed by dialysis. • Dialysis is the most common form of detergent removal and this process typically requires dialyzing the protein detergent mixtures against detergent-free buffer (in about 200-fold excess) over a period of days. If the protein is a membrane spanning protein, it may be denatured by detergent removal unless exogenous lipid is added. • This technique is more practical with detergents with a high cmc and works best for those with low molecular weight/small cross-sectional area. • The technique is unsuitable for detergents with a low cmc, for example, some of the nonionic detergents. • • Some detergents (e.g. octylglucoside, cholate) are easily removed by dilution ([ ] < CMC). These techniques permit the biochemist to mix exogenous lipid with detergent-solubilized integral membrane proteins and then to form sealed proteoliposomes by detergent removal using either dialysis or dilution/centrifugation. detergent removal + 47 Disadvantages of large aggregation number • ∆Gº for micelle formation from a long chain detergent is typically strongly negative (micelle formation is an exergonic reaction). These detergents form large micelles and have v low CMCs. • As a result, such detergents are often “nondialyzable” -they cannot be easily removed by dialysis or by dilution. • Such detergents are best removed using hydrophobic resins or beads (e.g. SM2) which have been developed to have low lipid and protein binding capacities. • This technique permits the biochemist to mix exogenous lipid with detergent solubilized integral membrane proteins and then to form sealed proteoliposomes by detergent removal using an appropriate resin. How does SDS denature proteins? • Tertiary structure unfolding at submicellar and chain expansion in the micellar range of SDS concentrations are two major and discrete events in the perturbation of protein structure. • The interaction between detergent and protein is predominantly hydrophobic in the submicellar and exclusively hydrophobic at micellar levels of SDS concentrations. • Expansion of the protein chain at micellar concentration of SDS is driven by ionic repulsion between the protein-bound micelles, micelles and anionic amino acid side chains. 48 Using CMT to our advantage • • • • Above the cmt, nonionic detergents become cloudy and phase separate into a detergent-rich layer and an aqueous layer. The temperature at which this occurs is called the cloud point. A low cloud point can be advantageous in the solubilization of membrane proteins, for example, the nonionic detergent Triton X-114 has a cloud point of 22ºC, thus the protein can be solubilized at 0ºC and then brought to 30ºC to allow phase separation to occur. The membrane protein will then partition into the detergent phase, which can then be separated by centrifugation. Can you think of any disadvantages of this methodology? Lipid Rafts In fact, these cholesterol rich domains are also highly enriched in sphingomyelin which is thought to preferentially associate with cholesterol due to their complementary geometries. 1 10 mL 5% sucrose Erythrocyte ghosts were mixed with ice-cold 2 2.5 ml of 1%(v/v) Triton X-100 in TBS and extracted on ice for 30 min. Extracts were 3 15 mL 35% sucrose mixed with 2.5 ml of 80% sucrose and 4 overlaid with 15 ml of 35% sucrose and 10 5 ml of 5% sucrose in TBS and 5 mL 40% sucrose 6 ultracentrifuged at 50,000g for 15 hr at 4ºC Solubilized RBC membranes allowing the various components in the mixture to distribute according to their buoyant density. The top 5 ml was collected as the first fraction. Proceeding down the gradient, fractions 2–6 were collected as 5 mL aliquots. Am. J. Hematol. 83:371–375, 2008 Rafts are thought to be involved in signal transduction although some scientists believe they are an artifact of isolation For a discussion on lipid rafts see: http://www.bms.ed.ac.uk/research/others/smaciver/Cyto-Topics/lipid_rafts_and_the_cytoskeleton.htm http://en.wikipedia.org/wiki/Lipid_raft Phospholipid distribution between hemileaflets of the lipid bilayer Lipid localization in biological membranes has been carried out primarily via chemical or enzymatic modification, via exchange techniques, and in some cases by immunochemical procedures. The digestion of phospholipids in the outer monolayer of a membrane by exogenous phospholipases may reveal the distribution of phospholipids between the two membrane layers. e.g. • • • • Phospholipase A2 - releases fatty acids from the second carbon group of glycerol Phospholipase C - cleaves phospholipids just before the phosphate group Phospholipases D - produces phosphatidic acid from phosphatidylcholine. Sphingomyelinase breaks SM into phosphocholine and ceramide. Lipid binding proteins (e.g. BSA) or lipid exchange vehicles (small unilamellar vesicles) can show which lipids, chemically modified lipids or enzymatically cleaved lipids are in the external hemileaflet. Phospholipid distribution between hemi-leaflets of the lipid bilayer external hemileaflet Total % of total phospholipid 50 Sphingomyelin Phosphatidylethanolamine 0 Phosphatidylcholine -50 Phosphatidylserine cytoplasmic hemileaflet • What effects could complicate this analysis? - observer effect. 52 Daleke, D. L. Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 2003;44:233-242. Today’s view of the cell membrane..... (“rafts” not included) The fluid mosaic model for membrane structure. The fatty acid chains in the interior of the membrane form a fluid, hydrophobic region. Integral membrane proteins float in this sea of lipid, held by hydrophobic interactions with their nonpolar amino acid side chains. Both proteins and lipids are free to move laterally in the plane of the bilayer, but movement of either from one face of the bilayer to the other is restricted. The carbohydrate moieties attached to some proteins and lipids of the plasma membrane are invariably exposed on the extracellular face of the membrane. Summary - Membranes 1. Lipids are amphipathic molecules with structurally diverse hydrophilic and hydrophobic domains 2. Lipid packing is determined by hydrophobic forces, lipid shape and lipid physical state 3. Phospholipids spontaneously assemble as bilayers in vitro and in vivo 4. Detergents are surfactants that solubilize membrane lipids and proteins. 5. Biomembranes display asymmetric lipid distributions between bilayer hemi-leaflets and may show lateral segregation of lipids within each hemi-leaflet 6. Bilayer-embedded lipids and proteins display high rates of lateral diffusion but low rates of flip flop