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UA BIOC 585 - BIOLOGICAL MEMBRANES

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PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59Slide 60Slide 61Slide 62Slide 63Slide 64Slide 65Slide 66Slide 67Slide 68Slide 69Slide 70Slide 71Slide 72Slide 73Slide 74Slide 75Slide 76Slide 77From Neutze et al. BBA 1565, 144 (2002)Slide 79Slide 80Slide 81Slide 82Slide 83Slide 84Slide 85Slide 86Slide 87Slide 88Slide 89Slide 90Slide 91BIOC. 585: BIOLOGICAL MEMBRANES! Overview biological rolesstructural features!Membrane lipidsgeneral structures aggregation states; polymorphismthermal transitions electrostatic effects molecular dynamics (translational and rotational diffusion, flip-flop) !Membrane proteinscrystallizationoverview of structural featuresstructure/function relations: photosynthetic electron transfer (cyt b6f)ion transport (K+ channels)bacteriorhodopsinREADING LISTBiochem. 585: Membrane Proteins Reading List [all articles in pdf files on course website]MEMBRANE PROTEIN CRYSTALLIZATIONOstermeier & Michel, “Crystallization of membrane proteins”, Curr. Op. Struct. Biol. 7, 697-701 (1997). Hunte & Michel, “Crystallization of membrane proteins mediated by antibody fragments”, Curr. Op.Struct. Biol. 12, 503-508 (2002).STRUCTURES AND FUNCTIONSOverviewScarlata, "Membrane Protein Structure", Chap. 1, Section 2, Biophysical Soc. on-line textbook.Byrne & Iwata, “Membrane protein complexes”, Curr. Opinion in Struct. Biol. 12, 239-243 (2002).Shipley, "Lipids; Bilayers and non-bilayers: structures, forces and protein crystallization", Curr. Op. Struct. Biol. 10, 471-473 (2000). Electron Transfer in PhotosynthesisOptional: Golbeck, “Photosynthetic Reaction Centers”, Biophysical Soc. on-line textbook.Kurisu et al., “Structure of the cytochrome b6f complex of oxygenic photosynthesis” Science 302, 1009-1014 (2003).Stroebel et al., “An atypical heme in the cytochrome b6f complex”, Nature 426, 413-418 (2003).Khlbrandt “Dual approach to a light problem”, Nature 426, 399-400 (2003)Ion ChannelsDoyle et al. “The structure of the potassium channel: molecular basis of K+ conduction and selectivity”, Science 280, 69-77 (1998).Rees et al. “Crystallographic analysis of ion channels: lessons and challenges” J. Biol. Chem. 275, 713-716 (2000).MacKinnon “Potassium channels” FEBS Letters 555, 62-65 (2003).Jiang et al. “X-ray structure of a voltage-dependent K+ channel”, Nature 423, 33-41 (2003). BacteriorhodopsinLanyi “Bacteriorhodopsin”, Bioenergetics, Chap. 3, Biophysical Soc. on-line textbook.Optional: Neutze et al. “Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport” Biochim. Biophys. Acta 1565, 144-167 (2002).BIOLOGICAL ROLES OF MEMBRANES selective permeability barriers (cell compartmentalization): pumps, gates, sieves structural organization of cellular processes (e.g. energy transduction): respiration, photosynthesis, vision receptors for external stimuli: hormones, neurotransmitters cell recognition: immune response, tissue formation intercellular communication: nerve impulse transmissionmost membranes are multi-functional~1/3 of all gene products in higher eukaryotes are membrane proteinsSTRUCTURAL FEATURES OF MEMBRANES MULTIPLE COMPONENTSlipids (phospholipids, glycolipids, cholesterol): bilayer structure forms main permeability barrier.proteins (peripheral, integral): provide both structural and functional characteristics.carbohydrate (covalently bound to lipid and protein): surface recognition. BROAD COMPOSITIONAL VARIABILITYcorrelated with function MOSTLY SELF ASSEMBLINGhydrophobic and electrostatic forces lead to bilayer formation and protein incorporation (carbohydrate added enzymically after assembly) ASYMMETRICinside different from outside with respect to lipid and protein (carbohydrate only found on outer surface) DYNAMIC STRUCTUREfluidity, flexibility, two-dimensional diffusionSTRUCTURES OF MEMBRANE LIPIDSLIPID POLYMORPHISMBIOLOGICAL SIGNIFICANCE OF LIPID POLYMORPHISM potential to form nonbilayer structures may allow discontinuities in bilayer and thereby promote:membrane fusion and vesicle formation during cell division.vesicle-mediated protein trafficking.integration of non-lipid components into membrane.transport of macromolecules through membrane.lateral movement of macromolecules.stabilization of membrane protein complexes.conformational interconversions associated with protein function.MEMBRANE DYNAMICSELECTROSTATICSTRANSLATIONAL (LATERAL) DIFFUSION IN MEMBRANES usually measured by FRAP (fluorescence recovery after photobleaching) using fluorophore-labelled lipids. Involves photobleaching a small region of membrane surface with laser and measuring time-dependence of molecular diffusion into bleached area. Dtrans (translational diffusion coefficient) related to mean square displacement: _r2  4 Dtrans t for both lipids and proteins, Dtrans  10-8 cm2s-1 at 25 °C. Thus, in 1 second: _ r2 = 4 x 10-8 cm2 _(r2)1/2 (mean displacement) = 2 x 10-4 cm = 2 microns (i.e. movement is rapid).MEASUREMENT OF MEMBRANE FLUIDITY AND MOLECULAR ROTATION BY FLUORESCENCE DEPOLARIZATIONuse a covalently attached fluorophore, or a fluorescent probe which partitions into the bilayer (e.g. DPH; diphenylhexatriene). Excite with polarized light and measure polarization of fluorescence. If fluorophore rotates during excited state lifetime, fluorescence will become depolarized.DEFINITIONS:P = polarization = (I - I) / (I + I) r = anisotropy = (I - I) / (I + 2I) PERRIN EQUATION:r0 / r = degree of depolarization = 1 + (F / C) WHERE:r0 = anisotropy in rigid matrix (i.e. no rotation)r = anisotropy in membrane F = fluorescence lifetime C = rotational correlation time = 1 / 6DrotDrot = rotational


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