MBIO 2100 14A Biomembranes 2024 PDF

25/10/24 MBIO 2100 Hypothesis: Lehninger Principles of Biochemistry The first cell originated when...

25/10/24 MBIO 2100 Hypothesis: Lehninger Principles of Biochemistry The first cell originated when a membrane enclosed a small 14A: Biological Membranes volume of aqueous solution. Text and figures from Lehninger 3rd - 7th ed., unless otherwise noted. Barbara H. Zimmermann, Ph.D. 1 2 Define external boundaries of cells, and regulate boundary traffic. Divide internal space into discrete compartments (eukaryotes) - segregation of metabolic processes and components. Central to energy conservation. Important in cell to cell communication. 3 4 PHYSICAL PROPERTIES Flexible Permit shape changes for growth and movement Self-sealing Can break and reseal, allows cell division or exocytosis Selectively permeable Impermeable to most polar or charged solutes, permeable to nonpolar compounds. Retain compounds Exclude compounds In electron microscopy, membranes appear Essentially two dimensional trilaminar: Composed of two layers of molecules (5 - 8 nm thick). Electron dense inner and outer surfaces Less dense core 5 6 1  25/10/24 Thin sections of a Lipids aggregate into structures in water Paramecium protozoa stained with osmium Aggregation reduces entropy of water tetratoxide. Structures formed depend on: (c) Mitochondrion Type and concentration of lipid (d) Digestive vacuole Three types of structures: (e) Endoplasmic reticulum Micelles Liposomes Bilayers 7 8 Amphipathic molecules Micelle hydrophilic hydrophobic Critical micelle concentration (CMC): concentration of molecules at which fatty acid aggregation occurs hydrophobic hydrophilic Number of lipid molecules in a micelle hundreds to thousands (Micela) sodium dodecyl sulfate (detergent) CMC of SDS is 8 mM, Triton X100 CMC = ≈ 0.23 mM 9 10 Vesicle (Liposome) Membrane Bilayer Small bilayers will Consists of two spontaneously seal into spherical vesicles leaflets (capas) of lipid monolayers Vesicles can contain artificially inserted Hydrophilic head proteins or enclose groups interact with dissolved molecules water (drugs) Hydrophobic fatty Vesicles fuse readily with (Vesícula) acid tails are packed cell membranes or with (Bicapa) each other inside 11 12 2  25/10/24 Study membrane composition, to understand membrane function. Which components are present in all membranes? Which components are unique to Myelin sheath of neurons membranes with specific functions? function: electrical insulation lipids > proteins Plasma membrane bacterium, mitochondrial membrane function: many enzyme catalyzed reactions proteins > lipids Values do not add up to 100% because not all lipids are listed 13 14 Cardiolipin Each type of membrane has characteristic lipids and proteins. Plasma: rich in cholesterol, no cardiolipin. Mitochondrial: less cholesterol, rich in cardiolipin. Cholesterol http://www.lipidhome.co.uk 15 16 Mito: inner/outer Lysosome 17 18 3  25/10/24 Erythrocyte plasma membrane ––––––––> Plasma membranes: asymmetric Each membrane has characteristic lipids and proteins. distributions of lipids Plasma: rich in cholesterol, no cardiolipin. in the outer and Mitochondrial: rich in cardiolipin, less cholesterol. inner monolayers. –> Cells control the kinds and amounts of lipids they synthesize. Not only do different membranes have different –> Cells target specific lipids to specific membranes. compositions, but also the two leaflets of the same membrane have different composition. 19 20 Changes in lipid distribution have biological consequences. example: In some cell types phosphatidylserine exposure on outer FLUID MOSAIC MODEL (1972) modelo del mosaico fluido: surface marks the cell (1) Fatty acyl chains in the interior of the membrane form a fluid for APOPTOSIS hydrophobic region. (=programmed cell death) (2) Integral membrane proteins (proteínas integrales de membrana) float in the sea of lipids. (3) Orientation of proteins in the bilayer is asymmetric. 21 22 FLUID MOSAIC MODEL (modelo del mosaico fluido): (4) Proteins and lipids move laterally in the plane of the bilayer. (5) Movement from one face to the other face of the (3) Orientation of proteins in the bilayer is asymmetric. membrane is restricted. (4) Proteins and lipids move laterally in plane of bilayer. 23 24 4  25/10/24 MEMBRANE PERIPHERAL PROTEIN ANCHORS Usually more than one lipid is necessary to anchor a protein. Glycosyl phosphatidyl inositol (GPI)- anchored Attachment of specific lipids target a protein to the correct side of the membrane. INTEGRAL PROTEINS 25 26 Types of Membrane Proteins How can we determine whether a protein is associated with a Amphitropic proteins: sometimes associated with membrane? membranes and sometimes not, depending on some type of regulatory process Analyze protein sequence to look such as reversible palmitoylation. for hydrophobic amino acids that might be associated with a membrane. 27 28 HYDROPATHY INDEX = A measure of the tendency of an amino acid to seek an aqueous environment. positive hydrophathy index: hydrophobic amino acids negative hydrophathy index: hydrophilic amino acids HYDROPATHY PLOTS (Representación de la hydrofobicidad) = Hydropathy index plotted against residue number. 29 30 5  25/10/24 How many segments of this protein cross the membrane? The presence of a continuous Cuantos segmentos de esta proteína atraviesan la membrana? sequence of 20 hydrophobic residues in a protein suggests that its polypeptide traverses a membrane. Genome analyses of different species suggests 10% -20% of coded proteins are associated with membranes. 31 32 Bacteriorhodopsin is Bacteriorhodopsin is an integral membrane an integral membrane protein whose crystal protein whose crystal structure has been structure has been determined. determined. Its polypeptide chain Its polypeptide chain forms 7 helices which forms 7 helices which span the membrane. span the membrane. Integral membrane protein structure: a-helices arranged in a cylinder. 33 34 Integral membrane protein structure: b-barrel Lys, Arg, Glu, Asp, are shown in blue. ≥ 20 transmembrane b-sheet segments Comparison of integral membrane proteins of known 7 - 9 residues are needed to span the membrane. crystal structure shows that tryptophan (red) and tyrosine (orange) residues cluster at the water-lipid interface. E. coli outer membrane proteins: FepA: iron uptake The R-groups of Trp and Tyr apparently are able to OmpLA: phopholipase (dimer) interact simultaneously with the lipid and aqueous phases. Maltoporin: maltose transporter (trimer) 35 36 6  25/10/24 MEMBRANE TOPOLOGY = the localization of the polypeptide chain relative to the lipid bilayer. How many ways can I insert this protein in a membrane? How can we determine what the membrane topology of a protein? TOPOLOGY 37 38 Determination of membrane topology: Experiment with glycophorin (131 residues), an integral protein of the erythrocyte membrane: 1. Incubate intact erythrocytes with trypsin. Trypsin cannot cross the membrane. (what is the activity of trypsin?) 2. Extract the protein from the erythrocyte and determine its size by denaturing gel electrophoresis. TOPOLOGY 39 40 1. protease OUTSIDE 2. 3. INSIDE 41 42 7  25/10/24 Determination of membrane topology: Glycophorin Experiment with glycophorin (131 residues), an One hydrophilic domain, integral protein of the erythrocyte membrane: containing sugar residues on the outer surface. RESULT: Glycophorin from trypsin-treated A second hydrophilic erythrocytes is smaller. It is ~ 60 residues shorter domain that protrudes from than the full length glycophorin. the inner surface of the membrane. CONCLUSION: ? A segment of 19 hydrophobic residues forms a helix that traverses the membrane. 43 44 Membrane bending proteins Caveolin A family of related protein. Have 3 palmitoyl anchors. Bind cholesterol. Form “rafts” involving both sides of bilayer. There are several different mechanisms for curving membranes 45 46 Membrane bending proteins Membrane bending proteins Caveolin CAVEOLIN FUNCTIONS: Involved in membrane trafficking within cell. Binds to inner layer of plasma membrane forcing Implicated in signal transduction (transmisión bilayer to curve (formation de señales biológicas). of vesicles). Expansion/contraction of cell. adipocyte membrane 47 48 8  25/10/24 Membrane fusion proteins Membrane fusion during neurotransmitter release at the synapse Membrane fusion requirements: 1. The surfaces recognize each other. 2. The surfaces become closely apposed. Water molecules associated with polar head groups are removed. 3. Outer leaflets of the two membranes fuse (hemifusion). 4. Single continuous bilayer forms. Fusion proteins (integral membrane proteins) mediate fusion. https://teachmephysiology.com/nervous-system/synapses/synaptic-transmission/ 49 50 Membrane fusion during neurotransmitter release at the synapse Membrane fusion during neurotransmitter release at the synapse https://teachmephysiology.com/nervous-system/synapses/synaptic-transmission/ 51 52 Membrane fusion during neurotransmitter release at the synapse The protease botulinum toxin from Clostridium botulinum attacks SNARE proteins and the SNAP 25 protein complex 53 54 9  25/10/24 Membrane fusion proteins Classification of Influenza A strains: H#N# H = hemagglutinin N = neuraminidase (=sialidase) The 2009 pandemic of “swine flu” was caused by H1N1 (0.2 – 0.6 million people died). In 1918, H1N1 influenza killed 20 – 50 million. So far ~ 7,000,000 have died from Covid-19 (Oct. 2023). Fusion induced by hemagglutinin (HA) protein during https://www.worldometers.info/coronavirus/coronavirus-death-toll/ viral infection. 55 56 Membrane Fusion MEMBRANE DYNAMICS Gel state (gel) : Polar head grous uniformly arrayed Membranes can fuse with each other at surface. without losing continuity Acyl chains nearly motionless, regularly packed. Fusion can be spontaneous or protein- mediated Liquid-ordered state : Less thermal motion in the acyl Examples of protein-mediated fusion chains. are: Lateral movement in the plane of – Entry of influenza virus into the host cell membrane can take place. Fluid state – Release of neurotransmitters at nerve Fluid state (= liquid-disordered state): synapses Acyl chains undergo much thermal motion and have no regular organization. 57 58 MEMBRANE DYNAMICS MEMBRANE DYNAMICS Gel state (gel) : Polar head grous uniformly arrayed at surface. Acyl chains nearly motionless, Cells require regularly packed. Biological Membranes membranes that have Liquid-ordered state : Less thermal motion in the acyl chains. an optimal fluidity. Lateral movement in the plane of membrane can take place. Fluid state Fluid state (= liquid-disordered state): Acyl chains undergo much thermal motion and have no regular organization. 59 60 10  25/10/24 MEMBRANE DYNAMICS MEMBRANE DYNAMICS Biological membranes are mixtures of lipids that Tight packing in fully saturated favor the liquid-orderd state. fatty acids. Lipid composition determines membrane fluidity: Less fluid. ––––––––> Unsaturated fatty acids have double bonds preventing tight packing (more fluid). Saturated fatty acids pack well into liquid-ordered Less ordered packing in arrays (less fluid). unsaturated fatty acids: Cholesterol’s rigid core reduces mobility of More fluid. –––––––> neighboring fatty acyl chains, forcing them into extended conformation. Cholesterol reduces membrane fluidity. 61 62 MEMBRANE DYNAMICS MEMBRANE DYNAMICS Would you expect the membrane composition of E. coli growing at 37°C to be the same as the membrane composition of E. coli growing at 25°C? Bacterial cells regulate their lipid composition to achieve constant fluidity under different growth conditions. 63 64 MEMBRANE DYNAMICS MEMBRANE DYNAMICS Lipid composition determines Cells require membranes membrane fluidity: that have an Unsaturated fatty acids optimal fluidity - more fluid Saturated fatty acids Biological membranses are - less fluid in a liquid-ordered state Cholesterol - less fluid 65 66 11  25/10/24 MEMBRANE DYNAMICS Transbilayer movement of lipids requires catalysis. To “ flip” polar head group: must leave the aqueous environment move through the hydrophobic Measuring the interior of the bilayer. Flippases movement of membrane transport lipids from inside surface of membrane to outside surface. lipids HOW DO WE MEASURE LATERAL MOVEMENT? 67 68 MEMBRANE DYNAMICS MEMBRANE DYNAMICS Lipids and proteins Lipids and proteins diffuse laterally in the diffuse laterally in the membrane. membrane. Lipid movement can be Lipid movement can be measured experimentally by measured experimentally by Fluorescence Recovery After Fluorescence Recovery After Photobleaching (FRAP). Photobleaching (FRAP). A molecule in the outer leaflet of A molecule in the outer leaflet of an erythrocyte membrane can an erythrocyte membrane can circumnavigate the cell in circumnavigate the cell in seconds. seconds. 69 70 Atomic Force Microscopy of Membrane proteins purified membrane proteins can be visualized by reconstituted in artificial membranes. Atomic-Force yellow = high above surface of Microscopy. membrane, closest to laser. brown = fartherest from to laser. Electrostatic interactions and van der Waals interactions between the probe tip Halobacterium salinarum and the membrane bacteriorhodopsin moves the probe up and down. E. coli aquaporin 71 72 12  25/10/24 The protein has inserted in the two possible orientations in the artificial membrane. What do cell membranes look like using atomic- top view (one of two) force microscopy? membrane chloroplast ATP synthase F0 subunit in an artificial side view Nakanishi et al. membrane Nat Commun. 2018;9:89. PMID: 29311594 73 74 Atomic force microscopy of a plasma The membrane is not uniform. membrane. Membrane microdomains Cluster of sphingolipids and cholesterol.The microdomains Liquid-ordered “rafts” in the “ocean” of the membrane. How would you interpret these results? ~ 50% of some cell surfaces are occupied by rafts. 75 76 “From the microbe’s point of view, lipid raft on the membrane of the host cell is an island with an airport in the middle of the ocean. Lipid rafts are characterized by the presence of a high concentration of outward looking protein molecules providing a guidance system and a landing gear to ensure precise and safe delivery of a microbe to the right terminal in a correct position. Lipid rafts host the endo- and exocytosis machineries – an Microdomains are slightly thicker than the surrounding automated loading/unloading facility connected to a railway effectively membrane, and are visible by atomic-force microscopy. transporting cargo to and from intracellular locations.” Proteins move in and out of the rafts on a time scale of seconds. viruses, intracellular bacteria, protozoa, fungi On the biochemically relevant time scale of microseconds, they remain in the rafts. Lipid rafts and pathogens: the art of deception and exploitation. Bukrinsky MI, Mukhamedova N, Sviridov D. J Lipid Res. 2020;61:601-610. doi: 10.1194/jlr.TR119000391. PMID: 31615838 77 78 13  25/10/24 MEMBRANE DYNAMICS MEMBRANE DYNAMICS Ordered regions (= lipid rafts) are shown in blue and green Disordered regions are shown in orange The plasma membranes of cells are composed of many types of Following stimulation two types of ordered membrane submicroscopic disordered (orange regions) and more ordered (all domains (or rafts) segregate to either pole of the cell, other regions) membrane domains,…. forming large assemblies (or flotillas) In resting leukocytes, all types of membrane domains… are evenly …In T cells, the flotilla at the front of the cell (blue region) is distributed around the cell periphery…” marked by the ganglioside GM3, whereas the flotilla at the rear (green region) contains the ganglioside GM1. Pierini, Lynda M. and Maxfield, Frederick R. (2001) Proc. Natl. Acad. Sci. USA 98, 9471-9473 Pierini, Lynda M. and Maxfield, Frederick R. (2001) Proc. Natl. Acad. Sci. USA 98, 9471-9473 Copyright ©2001 by the National Academy of Sciences Copyright ©2001 by the National Academy of Sciences 79 80 MEMBRANE DYNAMICS Single Particle Tracking MEMBRANE DYNAMICS Experiment to measure a single lipid molecule in a membrane. 1. Label a lipid with fluorescent tag. 2. Record movement on video by fluorescence microscopy at a resolution of 25 µs (40,000 frames per second!) FUNCTIONS OF RAFTS: example: chemotaxis of leukocytes Observation: Time lapse confocal microscopy of chemoattractant-stimulated leukocytes (Recording of 56 milliseconds) shows dynamic resdistribution of lipid rafts (green = raft components) Rapid diffusion occurs in a defined region, with occasional Gomez-Mouton et al., J Cell Biol. 2004;164:759-68. PMID: 14981096, Dynamic “hops” to another region. redistribution of raft domains as an organizing platform for signaling during cell chemotaxis. 81 82 MEMBRANE DYNAMICS Illustration of the “corral” effect of the membrane using Hexbugs in a Hexbug track (video). CONCLUSION: Lipids are “corralled” by molecular fences that they occasionally jump. When the hexbug is turned on, its Some membrane proteins have restricted movement because they The track is composed of 4 octagons. legs vibrate. are attached to spectrin, a cytoskeletal protein, or ankyrin Each octagon has small “doors” On/Off (examples: chloride-bicarbonate exchange protein, glycophorin). connecting it to a neighboring octagon. switch 83 84 14  25/10/24 85 15